Block copolymers for stable micelles

ABSTRACT

The present invention relates to the field of polymer chemistry and more particularly to multiblock copolymers and micelles comprising the same. Compositions herein are useful for drug-delivery applications.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of U.S. patent applicationSer. No. 14/694,760, filed Apr. 23, 2015, which claims priority to U.S.patent application Ser. No. 13/840,133, filed Mar. 15, 2013, whichclaims priority to U.S. provisional patent application Ser. No.61/622,755, filed Apr. 11, 2012, and U.S. provisional patent applicationSer. No. 61/659,841, filed Jun. 14, 2012, the entirety of each of whichare hereby incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the field of polymer chemistry and moreparticularly to multiblock copolymers and uses thereof.

BACKGROUND OF THE INVENTION

The development of new therapeutic agents has dramatically improved thequality of life and survival rate of patients suffering from a varietyof disorders. However, drug delivery innovations are needed to improvethe success rate of these treatments. Specifically, delivery systems arestill needed which effectively minimize premature excretion and/ormetabolism of therapeutic agents and deliver these agents specificallyto diseased cells thereby reducing their toxicity to healthy cells.

Rationally-designed, nanoscopic drug carriers, or “nanovectors,” offer apromising approach to achieving these goals due to their inherentability to overcome many biological barriers. Moreover, theirmulti-functionality permits the incorporation of cell-targeting groups,diagnostic agents, and a multitude of drugs in a single delivery system.Polymer micelles, formed by the molecular assembly of functional,amphiphilic block copolymers, represent one notable type ofmultifunctional nanovector.

Polymer micelles are particularly attractive due to their ability todeliver large payloads of a variety of drugs (e.g. small molecule,proteins, and DNA/RNA therapeutics), their improved in vivo stability ascompared to other colloidal carriers (e.g. liposomes), and theirnanoscopic size which allows for passive accumulation in diseasedtissues, such as solid tumors, by the enhanced permeation and retention(EPR) effect. Using appropriate surface functionality, polymer micellesare further decorated with cell-targeting groups and permeationenhancers that can actively target diseased cells and aid in cellularentry, resulting in improved cell-specific delivery.

While self assembly represents a convenient method for the bottom-updesign of nanovectors, the forces that drive and sustain the assembly ofpolymer micelles are concentration dependent and inherently reversible.In clinical applications, where polymer micelles are rapidly dilutedfollowing administration, this reversibility, along with highconcentrations of micelle-destabilizing blood components (e.g. proteins,lipids, and phospholipids), often leads to premature dissociation of thedrug-loaded micelle before active or passive targeting is effectivelyachieved. For polymer micelles to fully reach their cell-targetingpotential and exploit their envisioned multi-functionality, in vivocirculation time must be improved. Drug delivery vehicles are needed,which are infinitely stable to post-administration dilution, can avoidbiological barriers (e.g. reticuloendothelial system (RES) uptake), anddeliver drugs in response to the physiological environment encounteredin diseased tissues, such as solid tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic illustrations depicting the triblock copolymer (seeFIG. 1A) and polymer micelle (see FIG. 1B) of the present invention.

FIG. 2. Schematic illustrations showing the preparation of drug loadedmicelles.

FIG. 3. Schematic illustrations showing the crosslinking of a drugloaded micelle with metal ions.

FIG. 4. Schematic illustrations depicting the crosslinked, drug loadedmicelle of the present invention.

FIG. 5. Validation of encapsulation of daunorubicin by dialysis of theuncrosslinked formulation at 20 mg/ml (black bar) and 0.2 mg/mL (whitebar) for 6 hours against phosphate buffer pH 8.

FIG. 6. Iron-dependent crosslinking verification by dialysis at 0.2mg/mL in phosphate buffer pH 8 for 6 hours.

FIG. 7. Verification of time-dependency on iron-mediated crosslinking bydialysis at 0.2 mg/mL in phosphate buffer pH 8 for 6 hours.

FIG. 8. The uncrosslinked sample was reconstituted at 20 mg/ml and pHadjusted to 3, 4, 5, 6, 7, 7.4 and 8 to determine the pH dependency ofiron-mediated crosslinking. The samples were diluted to 0.2 mg/mL anddialyzed against 10 mM phosphate buffer pH 8 for 6 hours.

FIG. 9. pH-dependent release of crosslinked daunorubicin formulationdialyzed against 10 mM phosphate buffer pH adjusted to 3, 4, 5, 6, 7,7.4 and 8 for 6 hours.

FIG. 10. Salt-dependent release of the crosslinked daunorubicinformulation at 0.2 mg/mL dialyzed against 10 mM phosphate buffer withNaCl concentrations of 0, 10, 50, 100, 200, 300, 400 or 500 mM.

FIG. 11. DLS histogram demonstrating particle size distribution forcrosslinked aminopterin formulation.

FIG. 12. Verification of encapsulation by dialysis of the formulationabove (20 mg/mL, black bar) and below (0.2 mg/mL, white bar) the CMC.

FIG. 13. Verification of crosslinking and pH-dependent release ofaminopterin formulation at 0.2 mg/mL by dialysis in 10 mM phosphatebuffer over 6 hours.

FIG. 14. Cell viability for A549 lung cancer cells treated with freeaminopterin, uncrosslinked aminopterin formulation, crosslinkedaminopterin formulation, uncrosslinked empty micelle vehicle andcrosslinked empty micelle vehicle.

FIG. 15. Cell viability for OVCAR3 ovarian cancer cells treated withfree aminopterin, uncrosslinked aminopterin formulation, crosslinkedaminopterin formulation, uncrosslinked empty micelle vehicle andcrosslinked empty micelle vehicle.

FIG. 16. Cell viability for PANC-1 pancreatic (folate receptor+) cancercells treated with free aminopterin, uncrosslinked aminopterinformulation, crosslinked aminopterin formulation, uncrosslinked emptymicelle vehicle and crosslinked empty micelle vehicle.

FIG. 17. Cell viability for BxPC3 pancreatic (folate receptor−) cancercells treated with free aminopterin, uncrosslinked aminopterinformulation, crosslinked aminopterin formulation, uncrosslinked emptymicelle vehicle and crosslinked empty micelle vehicle.

FIG. 18. Concentration of SN-38 in the plasma compartment of rats fromIT-141 (NHOH; 127C) formulation compared to IT-141 (Asp; 127E)formulation at 10 mg/kg.

FIG. 19. Rat pharmacokinetics of SN-38 formulations.

FIG. 20. pH-dependent release of crosslinked cabizataxel formulationdialyzed against 10 mM phosphate buffer pH adjusted to 3, 4, 5, 6, 7,7.4 and 8 for 6 hours.

FIG. 21. Pharmacokinetics free daunorubicin and daunorubicinformulations in rats.

FIG. 22. Rat plasma levels of cabizataxel following administration ofcrosslinked cabizataxel formulation and free cabizataxel.

FIG. 23. Anti-tumor efficacy of crosslinked SN-38 formulations in anHCT-116 xenograft model.

FIG. 24. Biodistribution of aminopterin from crosslinked aminopterinformulations in an OVCAR-3 xenograft model.

FIG. 25. Anti-tumor efficacy of crosslinked aminopterin formulations inan MFE-296 xenograft model.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION 1. GeneralDescription

According to one embodiment, the present invention provides a micellecomprising a multiblock copolymer which comprises a polymerichydrophilic block, optionally a crosslinkable or crosslinked poly(aminoacid block), and a hydrophobic D,L-mixed poly(amino acid) block,characterized in that said micelle has an inner core, optionally acrosslinkable or crosslinked outer core, and a hydrophilic shell. Itwill be appreciated that the polymeric hydrophilic block corresponds tothe hydrophilic shell, the optionally crosslinkable or crosslinkedpoly(amino acid block) corresponds to the optionally crosslinked outercore, and the hydrophobic D,L-mixed poly(amino acid) block correspondsto the inner core.

The “hydrophobic D,L-mixed poly(amino acid)” block, as described herein,consists of a mixture of D and L enantiomers to facilitate theencapsulation of hydrophobic moieties. It is well established thathomopolymers and copolymers of amino acids, consisting of a singlestereoisomer, may exhibit secondary structures such as the α-helix orβ-sheet. See α-Aminoacid-N-Caroboxy-Anhydrides and Related Heterocycles,H. R. Kricheldorf, Springer-Verlag, 1987. For example, poly(L-benzylglutatmate) typically exhibits an α-helical conformation; however thissecondary structure can be disrupted by a change of solvent ortemperature (see Advances in Protein Chemistry XVI, P. Urnes and P.Doty, Academic Press, New York 1961). The secondary structure can alsobe disrupted by the incorporation of structurally dissimilar amino acidssuch as β-sheet forming amino acids (e.g. proline) or through theincorporation of amino acids with dissimilar stereochemistry (e.g.mixture of D and L stereoisomers), which results in poly(amino acids)with a random coil conformation. See Sakai, R.; Ikeda; S.; Isemura, T.Bull Chem. Soc. Japan 1969, 42, 1332-1336, Paolillo, L.; Temussi, P. A.;Bradbury, E. M.; Crane-Robinson, C. Biopolymers 1972, 11, 2043-2052, andCho, I.; Kim, J. B.; Jung, H. J. Polymer 2003, 44, 5497-5500.

While the methods to influence secondary structure of poly(amino acids)have been known for some time, it has been suprisingly discovered thatblock copolymers possessing a random coil conformation are particularlyuseful for the encapsulation of hydrophobic molecules and nanoparticleswhen compared to similar block copolymers possessing a helical segment.See US Patent Application 2008-0274173. Without wishing to be bound toany particular theory, it is believed that provided block copolymershaving a coil-coil conformation allow for efficient packing and loadingof hydrophobic moieties within the micelle core, while the stericdemands of a rod-coil conformation for a helix-containing blockcopolymer results in less effective encapsulation.

The hydrophobic forces that drive the aqueous assembly of colloidal drugcarriers, such as polymer micelles and liposomes, are relatively weak,and these assembled structures dissociate below a finite concentrationknown as the critical micelle concentration (CMC). The CMC value ofpolymer micelles is of great importance in clinical applications becausedrug-loaded colloidal carriers are diluted in the bloodstream followingadministration and rapidly reach concentrations below the CMC (μM orless). This dilution effect will lead to micelle dissociation and drugrelease outside the targeted area and any benefits associated with themicelle size (EPR effect) or active targeting will be lost. While agreat deal of research throughout the 1990's focused on identifyingpolymer micelles with ultra-low CMC values (nM or less), Maysinger(Savic et. al., Langmuir, 2006, p 3570-3578) and Schiochet (Lu et. al.,Macromolecules, 2011, p 6002-6008) have redefined the concept of abiologically relevant CMC by showing that the CMC values for polymermicelles shift by two orders of magnitude when the CMC values in salineare compared with and without serum.

In addition to their core-shell morphology, polymer micelles can bemodified to enable passive and active cell-targeting to maximize thebenefits of current and future therapeutic agents. Because drug-loadedmicelles typically possess diameters greater than 20 nm, they exhibitdramatically increased circulation time when compared to stand-alonedrugs due to minimized renal clearance. This unique feature ofnanovectors and polymeric drugs leads to selective accumulation indiseased tissue, especially cancerous tissue due to the enhancedpermeation and retention effect (“EPR”). The EPR effect is a consequenceof the disorganized nature of the tumor vasculature, which results inincreased permeability of polymer therapeutics and drug retention at thetumor site. In addition to passive cell targeting by the EPR effect,micelles are designed to actively target tumor cells through thechemical attachment of targeting groups to the micelle periphery. Theincorporation of such groups is most often accomplished throughend-group functionalization of the hydrophilic block using chemicalconjugation techniques. Like viral particles, micelles functionalizedwith targeting groups utilize receptor-ligand interactions to controlthe spatial distribution of the micelles after administration, furtherenhancing cell-specific delivery of therapeutics. In cancer therapy,targeting groups are designed to interact with receptors that areover-expressed in cancerous tissue relative to normal tissue such asfolic acid, oligopeptides, sugars, and monoclonal antibodies. See Pan,D.; Turner, J. L.; Wooley, K. L. Chem. Commun. 2003, 2400-2401; Gabizon,A.; Shmeeda, H.; Horowitz, A. T.; Zalipsky, S. Adv. Drug Deliv. Rev.2004, 56, 1177-1202; Reynolds, P. N.; Dmitriev, I.; Curiel, D. T.Vector. Gene Ther. 1999, 6, 1336-1339; Derycke, A. S. L.; Kamuhabwa, A.;Gijsens, A.; Roskams, T.; De Vos, D.; Kasran, A.; Huwyler, J.; Missiaen,L.; de Witte, P. A. M. T J. Nat. Cancer Inst. 2004, 96, 1620-30;Nasongkla, N., Shuai, X., Ai, H.; Weinberg, B. D. P., J.; Boothman, D.A.; Gao, J. Angew. Chem. Int. Ed. 2004, 43, 6323-6327; Jule, E.;Nagasaki, Y.; Kataoka, K. Bioconj. Chem. 2003, 14, 177-186; Stubenrauch,K.; Gleiter, S.; Brinkmann, U.; Rudolph, R.; Lilie, H. Biochem. J. 2001,356, 867-873; Kurschus, F. C.; Kleinschmidt, M.; Fellows, E.; Dornmair,K.; Rudolph, R.; Lilie, H.; Jenne, D. E. FEBS Lett. 2004, 562, 87-92;and Jones, S. D.; Marasco, W. A. Adv. Drug Del. Rev. 1998, 31, 153-170.

Despite the large volume of work on micellar drug carriers, littleeffort has focused on improving their in vivo stability to dilution. Onepotential reason is that the true effects of micelle dilution in vivoare not fully realized until larger animal studies are utilized. Becausea mouse's metabolism is much higher than larger animals, they canreceive considerably higher doses of toxic drugs when compared to largeranimals such as rats or dogs. Therefore, when drug loaded micelles areadministered and completely diluted throughout the entire blood volume,the corresponding polymer concentration will always be highest in themouse model. Therefore, it would be highly desirable to prepare amicelle that is stabilized (crosslinked) to dilution within biologicalmedia.

In the present invention, the optionally crosslinkable or crosslinkedpoly(amino acid block) is comprised of chemical functionality thatstrongly binds or coordinates with metal ions. One specific example ishydroxamic acids and iron (III). Another example is ortho-substituteddihydroxy benzene groups (catechols) with iron. Both hydroxamic acid andcatechol moieties are common in siderophores, high-affinity ironchelating agents produced by microorganisms. Additionally, it has beenreported that hydroxamic acid modified poly(acrylates) can form acrosslinked gel following treatment with iron (III) (Rosthauser andWinston, Macromolecules, 1981, p 538-543). Without wishing to be boundto any particular theory, it is believed that the incorporation of highaffinity metal chelating group such as hydroxamic acids and catechols inthe outer core of the micelle, following treatment with a metal ion willresult in a micelle that is stable to dilution within biological media.

Previous work has utilized carboxylic acids to interact with metal ionsin order to provide micelle stability. See US Patent Application2006-0240092. It has been surprisingly discovered that the use ofhydroxamic acid-modified polymers is effective at reversibly stabilizingthe polymer micelle to dilution within biological media. This hydroxamicacid chemistry has been demonstrated to be particularly effective whenencapsulating a drug that possesses one or more chemical functionalitiesknown to bind iron (e.g. carboxylic acids). Without wishing to be boundto any particular theory, it is believed that the metal ions used tostabilize the micelle will preferentially bind to the high affinitymetal chelating group such as hydroxamic acids and catechols, resultingin a stabilized micelle. Furthermore, the chelation reaction betweeniron (III) and hydroxamic acid moieties proceeds within seconds,allowing for a rapid crosslinking step.

2. Definitions

Compounds of this invention include those described generally above, andare further illustrated by the embodiments, sub-embodiments, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated. For purposes of this invention, the chemicalelements are identified in accordance with the Periodic Table of theElements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed.Additionally, general principles of organic chemistry are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5^(th) Ed.,Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.

As used herein, the term “sequential polymerization”, and variationsthereof, refers to the method wherein, after a first monomer (e.g. NCA,lactam, or imide) is incorporated into the polymer, thus forming anamino acid “block”, a second monomer (e.g. NCA, lactam, or imide) isadded to the reaction to form a second amino acid block, which processmay be continued in a similar fashion to introduce additional amino acidblocks into the resulting multi-block copolymers.

As used herein, the term “multiblock copolymer” refers to a polymercomprising one synthetic polymer portion and two or more poly(aminoacid) portions. Such multi-block copolymers include those having theformat W-X-X′, wherein W is a synthetic polymer portion and X and X′ arepoly(amino acid) chains or “amino acid blocks”. In certain embodiments,the multiblock copolymers of the present invention are triblockcopolymers. As described herein, one or more of the amino acid blocksmay be “mixed blocks”, meaning that these blocks can contain a mixtureof amino acid monomers thereby creating multiblock copolymers of thepresent invention. In some embodiments, the multiblock copolymers of thepresent invention comprise a mixed amino acid block and are tetrablockcopolymers.

One skilled in the art will recognize that a monomer repeat unit isdefined by parentheses around the repeating monomer unit. The number (orletter representing a numerical range) on the lower right of theparentheses represents the number of monomer units that are present inthe polymer chain. In the case where only one monomer represents theblock (e.g. a homopolymer), the block will be denoted solely by theparentheses. In the case of a mixed block, multiple monomers comprise asingle, continuous block. It will be understood that brackets willdefine a portion or block. For example, one block may consist of fourindividual monomers, each defined by their own individual set ofparentheses and number of repeat units present. All four sets ofparentheses will be enclosed by a set of brackets, denoting that allfour of these monomers combine in random, or near random, order tocomprise the mixed block. For clarity, the randomly mixed block of[BCADDCBADABCDABC] would be represented in shorthand by[(A)₄(B)₄(C)₄(D)₄].

As used herein, the monomer repeat unit described above is a numericalvalue representing the average number of monomer units comprising thepolymer chain. For example, a polymer represented by (A)₁₀ correspondsto a polymer consisting of ten “A” monomer units linked together. One ofordinary skill in the art will recognize that the number 10 in this casewill represent a distribution of numbers with an average of 10. Thebreadth of this distribution is represented by the polydispersity index(PDI). A PDI of 1.0 represents a polymer wherein each chain length isexactly the same (e.g. a protein). A PDI of 2.0 represents a polymerwherein the chain lengths have a Gaussian distribution. Polymers of thepresent invention typically possess a PDI of less than 1.20.

As used herein, the term “triblock copolymer” refers to a polymercomprising one synthetic polymer portion and two poly(amino acid)portions.

As used herein, the term “tetrablock copolymer” refers to a polymercomprising one synthetic polymer portion and either two poly(amino acid)portions, wherein 1 poly(amino acid) portion is a mixed block or apolymer comprising one synthetic polymer portion and three poly(aminoacid) portions.

As used herein, the term “inner core” as it applies to a micelle of thepresent invention refers to the center of the micelle formed by thehydrophobic D,L-mixed poly(amino acid) block. In accordance with thepresent invention, the inner core is not crosslinked. By way ofillustration, in a triblock polymer of the format W-X′-X″, as describedabove, the inner core corresponds to the X″ block.

As used herein, the term “outer core” as it applies to a micelle of thepresent invention refers to the layer formed by the first poly(aminoacid) block. The outer core lies between the inner core and thehydrophilic shell. In accordance with the present invention, the outercore is either crosslinkable or is cross-linked. By way of illustration,in a triblock polymer of the format W-X′-X″, as described above, theouter core corresponds to the X′ block. It is contemplated that the X′block can be a mixed block.

As used herein, the terms “drug-loaded” and “encapsulated”, andderivatives thereof, are used interchangeably. In accordance with thepresent invention, a “drug-loaded” micelle refers to a micelle having adrug, or therapeutic agent, situated within the core of the micelle. Incertain instances, the drug or therapeutic agent is situated at theinterface between the core and the hydrophilic coronoa. This is alsoreferred to as a drug, or therapeutic agent, being “encapsulated” withinthe micelle.

As used herein, the term “polymeric hydrophilic block” refers to apolymer that is not a poly(amino acid) and is hydrophilic in nature.Such hydrophilic polymers are well known in the art and includepolyethyleneoxide (also referred to as polyethylene glycol or PEG), andderivatives thereof, poly(N-vinyl-2-pyrolidone), and derivativesthereof, poly(N-isopropylacrylamide), and derivatives thereof,poly(hydroxyethyl acrylate), and derivatives thereof, poly(hydroxylethylmethacrylate), and derivatives thereof, and polymers ofN-(2-hydroxypropoyl)methacrylamide (HMPA) and derivatives thereof.

As used herein, the term “poly(amino acid)” or “amino acid block” refersto a covalently linked amino acid chain wherein each monomer is an aminoacid unit. Such amino acid units include natural and unnatural aminoacids. In certain embodiments, each amino acid unit of the optionallycrosslinkable or crosslinked poly(amino acid block) is in theL-configuration. Such poly(amino acids) include those having suitablyprotected functional groups. For example, amino acid monomers may havehydroxyl or amino moieties, which are optionally protected by a hydroxylprotecting group or an amine protecting group, as appropriate. Suchsuitable hydroxyl protecting groups and amine protecting groups aredescribed in more detail herein, infra. As used herein, an amino acidblock comprises one or more monomers or a set of two or more monomers.In certain embodiments, an amino acid block comprises one or moremonomers such that the overall block is hydrophilic. In still otherembodiments, amino acid blocks of the present invention include randomamino acid blocks, ie blocks comprising a mixture of amino acidresidues.

As used herein, the term “D,L-mixed poly(amino acid) block” refers to apoly(amino acid) block wherein the poly(amino acid) consists of amixture of amino acids in both the D- and L-configurations. In certainembodiments, the D,L-mixed poly(amino acid) block is hydrophobic. Inother embodiments, the D,L-mixed poly(amino acid) block consists of amixture of D-configured hydrophobic amino acids and L-configuredhydrophilic amino acid side-chain groups such that the overallpoly(amino acid) block comprising is hydrophobic.

Exemplary poly(amino acids) include poly(benzyl glutamate), poly(benzylaspartate), poly(L-leucine-co-tyrosine), poly(D-leucine-co-tyrosine),poly(L-phenylalanine-co-tyrosine), poly(D-phenylalanine-co-tyrosine),poly(L-leucine-coaspartic acid), poly(D-leucine-co-aspartic acid),poly(L-phenylalanine-co-aspartic acid), poly(D-phenylalanine-co-asparticacid).

As used herein, the phrase “natural amino acid side-chain group” refersto the side-chain group of any of the 20 amino acids naturally occurringin proteins. For clarity, the side chain group —CH₃ would represent theamino acid alanine. Such natural amino acids include the nonpolar, orhydrophobic amino acids, glycine, alanine, valine, leucine isoleucine,methionine, phenylalanine, tryptophan, and proline. Cysteine issometimes classified as nonpolar or hydrophobic and other times aspolar. Natural amino acids also include polar, or hydrophilic aminoacids, such as tyrosine, serine, threonine, aspartic acid (also known asaspartate, when charged), glutamic acid (also known as glutamate, whencharged), asparagine, and glutamine. Certain polar, or hydrophilic,amino acids have charged side-chains. Such charged amino acids includelysine, arginine, and histidine. One of ordinary skill in the art wouldrecognize that protection of a polar or hydrophilic amino acidside-chain can render that amino acid nonpolar. For example, a suitablyprotected tyrosine hydroxyl group can render that tyroine nonpolar andhydrophobic by virtue of protecting the hydroxyl group.

As used herein, the phrase “unnatural amino acid side-chain group”refers to amino acids not included in the list of 20 amino acidsnaturally occurring in proteins, as described above. Such amino acidsinclude the D-isomer of any of the 20 naturally occurring amino acids.Unnatural amino acids also include homoserine, ornithine, and thyroxine.Other unnatural amino acids side-chains are well know to one of ordinaryskill in the art and include unnatural aliphatic side chains. Otherunnatural amino acids include modified amino acids, including those thatare N-alkylated, cyclized, phosphorylated, acetylated, amidated,azidylated, labelled, and the like.

As used herein, the term “tacticity” refers to the stereochemistry ofthe poly(amino acid) hydrophobic block. A poly(amino acid) blockconsisting of a single stereoisomer (e.g. all L isomer) is referred toas “isotactic”. A poly(amino acid) consisting of a random incorporationof D and L amino acid monomers is referred to as an “atactic” polymer. Apoly(amino acid) with alternating stereochemistry (e.g. . . . DLDLDL . .. ) is referred to as a “syndiotactic” polymer. Polymer tacticity isdescribed in more detail in “Principles of Polymerization”, 3rd Ed., G.Odian, John Wiley & Sons, New York: 1991, the entire contents of whichare hereby incorporated by reference.

As used herein, the phrase “living polymer chain-end” refers to theterminus resulting from a polymerization reaction which maintains theability to react further with additional monomer or with apolymerization terminator.

As used herein, the term “termination” refers to attaching a terminalgroup to a polymer chain-end by the reaction of a living polymer with anappropriate compound. Alternatively, the term “termination” may refer toattaching a terminal group to an amine or hydroxyl end, or derivativethereof, of the polymer chain.

As used herein, the term “polymerization terminator” is usedinterchangeably with the term “polymerization terminating agent” andrefers to a compound that reacts with a living polymer chain-end toafford a polymer with a terminal group. Alternatively, the term“polymerization terminator” may refer to a compound that reacts with anamine or hydroxyl end, or derivative thereof, of the polymer chain, toafford a polymer with a terminal group.

As used herein, the term “polymerization initiator” refers to acompound, which reacts with, or whose anion or free base form reactswith, the desired monomer in a manner which results in polymerization ofthat monomer. In certain embodiments, the polymerization initiator isthe compound that reacts with an alkylene oxide to afford a polyalkyleneoxide block. In other embodiments, the polymerization initiator is anamine salt as described herein. In certain embodiments, thepolymerization initiator is a trifluoroacetic acid amine salt.

The term “aliphatic” or “aliphatic group”, as used herein, denotes ahydrocarbon moiety that may be straight-chain (i.e., unbranched),branched, or cyclic (including fused, bridging, and spiro-fusedpolycyclic) and may be completely saturated or may contain one or moreunits of unsaturation, but which is not aromatic. Unless otherwisespecified, aliphatic groups contain 1-20 carbon atoms. In someembodiments, aliphatic groups contain 1-10 carbon atoms. In otherembodiments, aliphatic groups contain 1-8 carbon atoms. In still otherembodiments, aliphatic groups contain 1-6 carbon atoms, and in yet otherembodiments aliphatic groups contain 1-4 carbon atoms. Aliphatic groupsinclude, but are not limited to, linear or branched, alkyl, alkenyl, andalkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl,(cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,phosphorus, or silicon. This includes any oxidized form of nitrogen,sulfur, phosphorus, or silicon; the quaternized form of any basicnitrogen, or; a substitutable nitrogen of a heterocyclic ring including═N— as in 3,4-dihydro-2H-pyrrolyl, —NH— as in pyrrolidinyl, or═N(R^(†))— as in N— substituted pyrrolidinyl.

The term “unsaturated”, as used herein, means that a moiety has one ormore units of unsaturation.

As used herein, the term “bivalent, saturated or unsaturated, straightor branched C₁₋₁₂ hydrocarbon chain”, refers to bivalent alkylene,alkenylene, and alkynylene chains that are straight or branched asdefined herein.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic,bicyclic, and tricyclic ring systems having a total of five to fourteenring members, wherein at least one ring in the system is aromatic andwherein each ring in the system contains three to seven ring members.The term “aryl” may be used interchangeably with the term “aryl ring”.

As described herein, compounds of the invention may contain “optionallysubstituted” moieties. In general, the term “substituted”, whetherpreceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substituted”group may have a suitable substituent at each substitutable position ofthe group, and when more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds. The term “stable”, as used herein, refers tocompounds that are not substantially altered when subjected toconditions to allow for their production, detection, and, in certainembodiments, their recovery, purification, and use for one or more ofthe purposes disclosed herein.

Monovalent substituents on a substitutable carbon atom of an “optionallysubstituted” group are independently halogen; —(CH₂)₀₋₄R^(∘);—(CH₂)₀₋₄OR^(∘); —O—(CH₂)₀₋₄C(O)OR^(∘); —(CH₂)₀₋₄CH(OR^(∘))₂;—(CH₂)₀₋₄SR^(∘); —(CH₂)₀₋₄Ph, which may be substituted with R^(∘);—(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substituted with R^(∘); —CH═CHPh,which may be substituted with R^(∘); —NO₂; —CN; —N₃; —(CH₂)₀₋₄N(R^(∘))₂;—(CH₂)₀₋₄N(R^(∘))C(O)R^(∘); —N(R^(∘))C(S)R^(∘); —(CH₂)₀₋₄N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))C(S)NR^(∘) ₂;—(CH₂)₀₋₄N(R^(∘))C(O)OR^(∘); —N(R^(∘))N(R^(∘))C(O)R^(∘);—N(R^(∘))N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))N(R^(∘))C(O)OR^(∘);—(CH₂)₀₋₄C(O)R^(∘); —C(S)R^(∘); —(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄C(O)SR^(∘); —(CH₂)₀₋₄C(O)OSiR^(∘) ₃; —(CH₂)₀₋₄C(O)R^(∘);—OC(O)(CH₂)₀₋₄SR—, SC(S)SR^(∘); —(CH₂)₀₋₄SC(O)R^(∘); —(CH₂)₀₋₄C(O)NR^(∘)₂; —C(S)NR^(∘) ₂; —C(S)SR^(∘); —SC(S)SR^(∘)), —(CH₂)₀₋₄OC(O)NR^(∘) ₂;—C(O)N(OR^(∘))R^(∘); —C(O)C(O)R^(∘); —C(O)CH₂C(O)R^(∘);—C(NOR^(∘))R^(∘); —(CH₂)₀₋₄SSR^(∘); —(CH₂)₀₋₄S(O)₂R^(∘);—(CH₂)₀₋₄S(O)₂OR^(∘); —(CH₂)₀₋₄OS(O)₂R^(∘); —S(O)₂NR^(∘) ₂;—(CH₂)₀₋₄S(O)R^(∘); —N(R^(∘))S(O)₂NR^(∘) ₂; —N(R^(∘))S(O)₂R^(∘);—N(OR^(∘))R^(∘); —C(NH)NR^(∘) ₂; —P(O)₂R^(∘); —P(O)R^(∘) ₂; —OP(O)R^(∘)₂; —OP(O)(OR^(∘))₂; SiR^(∘) ₃; —(C₁₋₄ straight or branchedalkylene)O—N(R^(∘))₂; or —(C₁₋₄ straight or branchedalkylene)C(O)O—N(R^(∘))₂, wherein each R^(∘) may be substituted asdefined below and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, or, notwithstanding the definition above, twoindependent occurrences of R^(∘), taken together with their interveningatom(s), form a 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur, which may be substituted as definedbelow.

Monovalent substituents on R^(∘))(or the ring formed by taking twoindependent occurrences of R^(∘) together with their intervening atoms),are independently halogen, —(CH₂)₀₋₂R^(●), -(haloR^(●)), —(CH₂)₀₋₂OH,—(CH₂)₀₋₂OR^(●), —(CH₂)₀₋₂CH(OR^(●))₂; —O(haloR^(●)), —CN, —N₃,—(CH₂)₀₋₂C(O)R^(●), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)O^(●),—(CH₂)₀₋₂SR^(●), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(●),—(CH₂)₀₋₂NR^(●) ₂, —NO₂, —SiR^(●) ₃, —OSiR^(●) ₃, —C(O)SR^(●), —(C₁₋₄straight or branched alkylene)C(O)OR^(●), or —SSR^(●) wherein each R^(●)is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Such divalent substituents on asaturated carbon atom of R^(∘) include ═O and ═S.

Divalent substituents on a saturated carbon atom of an “optionallysubstituted” group include the following: ═O, ═S, ═NNR*₂, ═NNHC(O)R*,═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Divalent substituents that are bound to vicinalsubstitutable carbons of an “optionally substituted” group include:—O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. A tetravalent substituent that is bound to vicinalsubstitutable methylene carbons of an “optionally substituted” group isthe dicobalt hexacarbonyl cluster represented by

when depicted with the methylenes which bear it.

Suitable substituents on the aliphatic group of R* include halogen,—R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN, —C(O)OH,—C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein each R^(●) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂, —C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein each R^(†) isindependently hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN,—C(O)OH, —C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein eachR^(●) is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Protected hydroxyl groups are well known in the art and include thosedescribed in detail in Protecting Groups in Organic Synthesis, T. W.Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999, theentirety of which is incorporated herein by reference. Examples ofsuitably protected hydroxyl groups further include, but are not limitedto, esters, carbonates, sulfonates allyl ethers, ethers, silyl ethers,alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples ofsuitable esters include formates, acetates, proprionates, pentanoates,crotonates, and benzoates. Specific examples of suitable esters includeformate, benzoyl formate, chloroacetate, trifluoroacetate,methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate,pivaloate (trimethylacetate), crotonate, 4-methoxy-crotonate, benzoate,p-benylbenzoate, 2,4,6-trimethylbenzoate. Examples of carbonates include9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl,2-(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl carbonate.Examples of silyl ethers include trimethylsilyl, triethylsilyl,t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl ether, andother trialkylsilyl ethers. Examples of alkyl ethers include methyl,benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and allylether, or derivatives thereof. Alkoxyalkyl ethers include acetals suchas methoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl,benzyloxymethyl, beta-(trimethylsilyl)ethoxymethyl, andtetrahydropyran-2-yl ether. Examples of arylalkyl ethers include benzyl,p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and4-picolyl ethers.

Protected amines are well known in the art and include those describedin detail in Greene (1999). Mono-protected amines further include, butare not limited to, aralkylamines, carbamates, allyl amines, amides, andthe like. Examples of mono-protected amino moieties includet-butyloxycarbonylamino (—NHBOC), ethyloxycarbonylamino,methyloxycarbonylamino, trichloroethyloxycarbonylamino,allyloxycarbonylamino (—NHAlloc), benzyloxocarbonylamino (—NHCBZ),allylamino, benzylamino (—NHBn), fluorenylmethylcarbonyl (—NHFmoc),formamido, acetamido, chloroacetamido, dichloroacetamido,trichloroacetamido, phenylacetamido, trifluoroacetamido, benzamido,t-butyldiphenylsilyl, and the like. Di-protected amines include aminesthat are substituted with two substituents independently selected fromthose described above as mono-protected amines, and further includecyclic imides, such as phthalimide, maleimide, succinimide, and thelike. Di-protected amines also include pyrroles and the like,2,2,5,5-tetramethyl-[1,2,5]azadisilolidine and the like, and azide.

Protected aldehydes are well known in the art and include thosedescribed in detail in Greene (1999). Protected aldehydes furtherinclude, but are not limited to, acyclic acetals, cyclic acetals,hydrazones, imines, and the like. Examples of such groups includedimethyl acetal, diethyl acetal, diisopropyl acetal, dibenzyl acetal,bis(2-nitrobenzyl) acetal, 1,3-dioxanes, 1,3-dioxolanes, semicarbazones,and derivatives thereof.

Protected carboxylic acids are well known in the art and include thosedescribed in detail in Greene (1999). Protected carboxylic acids furtherinclude, but are not limited to, optionally substituted C₁₋₆ aliphaticesters, optionally substituted aryl esters, silyl esters, activatedesters, amides, hydrazides, and the like. Examples of such ester groupsinclude methyl, ethyl, propyl, isopropyl, butyl, isobutyl, benzyl, andphenyl ester, wherein each group is optionally substituted. Additionalprotected carboxylic acids include oxazolines and ortho esters.

Protected thiols are well known in the art and include those describedin detail in Greene (1999). Protected thiols further include, but arenot limited to, disulfides, thioethers, silyl thioethers, thioesters,thiocarbonates, and thiocarbamates, and the like. Examples of suchgroups include, but are not limited to, alkyl thioethers, benzyl andsubstituted benzyl thioethers, triphenylmethyl thioethers, andtrichloroethoxycarbonyl thioester, to name but a few.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, Z and E double bond isomers,and Z and E conformational isomers. Therefore, single stereochemicalisomers as well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds are within the scopeof the invention. Unless otherwise stated, all tautomeric forms of thecompounds of the invention are within the scope of the invention.Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures except for the replacement of hydrogen by deuteriumor tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enrichedcarbon are within the scope of this invention. Such compounds areuseful, for example, as in neutron scattering experiments, as analyticaltools or probes in biological assays.

As used herein, the term “detectable moiety” is used interchangeablywith the term “label” and relates to any moiety capable of beingdetected (e.g., primary labels and secondary labels). A “detectablemoiety” or “label” is the radical of a detectable compound.

“Primary” labels include radioisotope-containing moieties (e.g.,moieties that contain ³²P, ³³P, ³⁵S, or ¹⁴C), mass-tags, and fluorescentlabels, and are signal-generating reporter groups which can be detectedwithout further modifications.

Other primary labels include those useful for positron emissiontomography including molecules containing radioisotopes (e.g. ¹⁸F) orligands with bound radioactive metals (e.g. ⁶²Cu). In other embodiments,primary labels are contrast agents for magnetic resonance imaging suchas gadolinium, gadolinium chelates, or iron oxide (e.g Fe₃O₄ and Fe₂O₃)particles. Similarly, semiconducting nanoparticles (e.g. cadmiumselenide, cadmium sulfide, cadmium telluride) are useful as fluorescentlabels. Other metal nanoparticles (e.g colloidal gold) also serve asprimary labels.

“Secondary” labels include moieties such as biotin, or protein antigens,that require the presence of a second compound to produce a detectablesignal. For example, in the case of a biotin label, the second compoundmay include streptavidin-enzyme conjugates. In the case of an antigenlabel, the second compound may include an antibody-enzyme conjugate.Additionally, certain fluorescent groups can act as secondary labels bytransferring energy to another compound or group in a process ofnonradiative fluorescent resonance energy transfer (FRET), causing thesecond compound or group to then generate the signal that is detected.

Unless otherwise indicated, radioisotope-containing moieties areoptionally substituted hydrocarbon groups that contain at least oneradioisotope. Unless otherwise indicated, radioisotope-containingmoieties contain from 1-40 carbon atoms and one radioisotope. In certainembodiments, radioisotope-containing moieties contain from 1-20 carbonatoms and one radioisotope.

The terms “fluorescent label”, “fluorescent group”, “fluorescentcompound”, “fluorescent dye”, and “fluorophore”, as used herein, referto compounds or moieties that absorb light energy at a definedexcitation wavelength and emit light energy at a different wavelength.Examples of fluorescent compounds include, but are not limited to: AlexaFluor dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, AlexaFluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, AlexaFluor 660 and Alexa Fluor 680), AMCA, AMCA-S, BODIPY dyes (BODIPY FL,BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568,BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY650/665), Carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Cascade Blue,Cascade Yellow, Coumarin 343, Cyanine dyes (Cy3, Cy5, Cy3.5, Cy5.5),Dansyl, Dapoxyl, Dialkylaminocoumarin,4′,5′-Dichloro-2′,7′-dimethoxy-fluorescein, DM-NERF, Eosin, Erythrosin,Fluorescein, FAM, Hydroxycoumarin, IRDyes (IRD40, IRD 700, IRD 800),JOE, Lissamine rhodamine B, Marina Blue, Methoxycoumarin,Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, PyMPO, Pyrene, Rhodamine B, Rhodamine 6G, RhodamineGreen, Rhodamine Red, Rhodol Green,2′,4′,5′,7′-Tetra-bromosulfone-fluorescein, Tetramethyl-rhodamine (TMR),Carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X.

The term “substrate”, as used herein refers to any material ormacromolecular complex to which a functionalized end-group of a blockcopolymer can be attached. Examples of commonly used substrates include,but are not limited to, glass surfaces, silica surfaces, plasticsurfaces, metal surfaces, surfaces containing a metalic or chemicalcoating, membranes (e.g., nylon, polysulfone, silica), micro-beads(e.g., latex, polystyrene, or other polymer), porous polymer matrices(e.g., polyacrylamide gel, polysaccharide, polymethacrylate),macromolecular complexes (e.g., protein, polysaccharide).

The term hydroxamic acid, as used herein, refers to a moiety containinga hydroxamic acid (—CO—NH—OH) functional group. The structured isrepresented by

and may also be represented by

One skilled in the art would recognize that the dotted bond representsthe attachment point to the rest of the molecule.

The term hydroxamate, as used herein, refers to a moiety containingeither hydroxamic acid or an N-substituted hydroxamic acid. Due to theN-substitution, two separate locations exist for chemical attachment, asshown by the R and R′ groups here

Hydoxamates may also be represented by

herein.

The term catechol, as used herein, refers to a substitutedortho-dihydroxybenezene derivative. Two different isomeric conformationsare represented by

Catechol is also known as pyrocatechol and benzene-1,2-diol.

3. Description of Exemplary Embodiments

A. Multiblock Copolymers

In certain embodiments, the multiblock copolymer comprises a hydrophilicpoly(ethylene glycol) block, a hydroxamic acid-containing poly(aminoacid) block, and a hydrophobic poly(amino acid) block characterized inthat the resulting micelle has an inner core, a hydroxamicacid-containing outer core, and a hydrophilic shell. It will beappreciated that the hydrophilic poly(ethylene glycol) block correspondsto the hydrophilic shell, stabilizing hydroxamic acid-containingpoly(amino acid) block corresponds to the hydroxamic acid-containingouter core, and the hydrophobic poly(amino acid) block corresponds tothe inner core.

In other embodiments, the multiblock copolymer comprises a hydrophilicpoly(ethylene glycol) block, a catechol-containing poly(amino acid)block, and a hydrophobic poly(amino acid) block characterized in thatthe resulting micelle has an inner core, an catechol-containing outercore, and a hydrophilic shell. It will be appreciated that thehydrophilic poly(ethylene glycol) block corresponds to the hydrophilicshell, stabilizing catechol-containing poly(amino acid) blockcorresponds to the catechol-containing outer core, and the hydrophobicpoly(amino acid) block corresponds to the inner core.

In certain embodiments, the multiblock copolymer comprises a hydrophilicpoly(ethylene glycol) block, a hydroxamate-containing poly(amino acid)block, and a hydrophobic poly(amino acid) block characterized in thatthe resulting micelle has an inner core, a hydroxamate-containing outercore, and a hydrophilic shell. It will be appreciated that thehydrophilic poly(ethylene glycol) block corresponds to the hydrophilicshell, stabilizing hydroxamate-containing poly(amino acid) blockcorresponds to the hydroxamate-containing outer core, and thehydrophobic poly(amino acid) block corresponds to the inner core.

In certain embodiments, the multiblock copolymer comprises a hydrophilicpoly(ethylene glycol) block, a hydroxamic acid-containing poly(aminoacid) block, and a hydrophobic D,L mixed poly(amino acid) blockcharacterized in that the resulting micelle has an inner core, ahydroxamic acid-containing outer core, and a hydrophilic shell. It willbe appreciated that the hydrophilic poly(ethylene glycol) blockcorresponds to the hydrophilic shell, stabilizing hydroxamicacid-containing poly(amino acid) block corresponds to the hydroxamicacid-containing outer core, and the hydrophobic D,L mixed poly(aminoacid) block corresponds to the inner core.

In other embodiments, the multiblock copolymer comprises a hydrophilicpoly(ethylene glycol) block, a catechol-containing poly(amino acid)block, and a hydrophobic D,L mixed poly(amino acid) block characterizedin that the resulting micelle has an inner core, an catechol-containingouter core, and a hydrophilic shell. It will be appreciated that thehydrophilic poly(ethylene glycol) block corresponds to the hydrophilicshell, stabilizing catechol-containing poly(amino acid) blockcorresponds to the catechol-containing outer core, and the hydrophobicD,L mixed poly(amino acid) block corresponds to the inner core.

In certain embodiments, the multiblock copolymer comprises a hydrophilicpoly(ethylene glycol) block, a hydroxamate-containing poly(amino acid)block, and a hydrophobic D,L mixed poly(amino acid) block characterizedin that the resulting micelle has an inner core, ahydroxamate-containing outer core, and a hydrophilic shell. It will beappreciated that the hydrophilic poly(ethylene glycol) block correspondsto the hydrophilic shell, stabilizing hydroxamate-containing poly(aminoacid) block corresponds to the hydroxamate-containing outer core, andthe hydrophobic D,L mixed poly(amino acid) block corresponds to theinner core.

In certain embodiments, the present invention provides a triblockcopolymer of formula I:

wherein:

-   -   n is 20-500;    -   x is 3 to 50;    -   y is 5 to 100;    -   R^(x) is a hydroxamate or catechol containing moiety;    -   R^(y) is selected from one or more natural or unnatural amino        acid side chain groups such that the overall block is        hydrophobic;    -   R¹ is —Z(CH₂CH₂Y)_(p)(CH₂)_(t)R³, wherein:        -   Z is —O—, —NH—, —S—, —C≡C—, or —CH₂—;        -   each Y is independently —O— or —S—;        -   p is 0-10;        -   t is 0-10; and    -   R³ is hydrogen, —N₃, —CN, —NH₂, —CH₃,

-   -    a strained cyclooctyne moiety, a mono-protected amine, a        di-protected amine, an optionally protected aldehyde, an        optionally protected hydroxyl, an optionally protected        carboxylic acid, an optionally protected thiol, or an optionally        substituted group selected from aliphatic, a 5-8 membered        saturated, partially unsaturated, or aryl ring having 0-4        heteroatoms independently selected from nitrogen, oxygen, or        sulfur, an 8-10 membered saturated, partially unsaturated, or        aryl bicyclic ring having 0-5 heteroatoms independently selected        from nitrogen, oxygen, or sulfur, or a detectable moiety;    -   Q is a valence bond or a bivalent, saturated or unsaturated,        straight or branched C₁₋₁₂ hydrocarbon chain, wherein 0-6        methylene units of Q are independently replaced by -Cy-, —O—,        —NH—, —S—, —OC(O)—, —C(O)O—, —C(O)—, —SO—, —SO₂—, —NHSO₂—,        —SO₂NH—, —NHC(O)—, —C(O)NH—, —OC(O)NH—, or —NHC(O)O—, wherein:        -   -Cy- is an optionally substituted 5-8 membered bivalent,            saturated, partially unsaturated, or aryl ring having 0-4            heteroatoms independently selected from nitrogen, oxygen, or            sulfur, or an optionally substituted 8-10 membered bivalent            saturated, partially unsaturated, or aryl bicyclic ring            having 0-5 heteroatoms independently selected from nitrogen,            oxygen, or sulfur;    -   R² is a mono-protected amine, a di-protected amine, N(R⁴)₂,        —NR⁴C(O)R⁴, —NR⁴C(O)N(R⁴)₂, —NR⁴C(O)OR⁴, or —NR⁴SO₂R⁴; and    -   each R⁴ is independently hydrogen or an optionally substituted        group selected from aliphatic, a 5-8 membered saturated,        partially unsaturated, or aryl ring having 0-4 heteroatoms        independently selected from nitrogen, oxygen, or sulfur, an 8-10        membered saturated, partially unsaturated, or aryl bicyclic ring        having 0-5 heteroatoms independently selected from nitrogen,        oxygen, or sulfur, or a detectable moiety, or:        -   two R⁴ on the same nitrogen atom are taken together with            said nitrogen atom to form an optionally substituted 4-7            membered saturated, partially unsaturated, or aryl ring            having 1-4 heteroatoms independently selected from nitrogen,            oxygen, or sulfur.

According to another embodiment, the present invention providescompounds of formula I, as described above, wherein said compounds havea polydispersity index (“PDI”) of 1.0 to 1.2. According to anotherembodiment, the present invention provides compounds of formula I, asdescribed above, wherein said compound has a polydispersity index(“PDI”) of 1.01 to 1.10. According to yet another embodiment, thepresent invention provides compounds of formula I, as described above,wherein said compound has a polydispersity index (“PDI”) of 1.10 to1.20. According to other embodiments, the present invention providescompounds of formula I having a PDI of less than 1.10.

As defined generally above, the n is 20 to 500. In certain embodiments,the present invention provides compounds wherein n is 225. In otherembodiments, n is 40 to 60. In other embodiments, n is 60 to 90. Instill other embodiments, n is 90 to 150. In other embodiments, n is 150to 200. In some embodiments, n is 200 to 300, 300 to 400, or 400 to 500.In still other embodiments, n is 250 to 280. In other embodiments, n is300 to 375. In other embodiments, n is 400 to 500. In certainembodiments, n is selected from 50±10. In other embodiments, n isselected from 80±10, 115±10, 180±10, 225±10, or 275±10.

In certain embodiments, the x is 3 to 50. In certain embodiments, the xis 10. In other embodiments, x is 20. According to yet anotherembodiment, x is 15. In other embodiments, x is 5. In other embodiments,x is selected from 5±3, 10±3, 10±5, 15±5, or 20±5.

In certain embodiments, y is 5 to 100. In certain embodiments, y is 10.In other embodiments, y is 20. According to yet another embodiment, y is15. In other embodiments, y is 30. In other embodiments, y is selectedfrom 10±3, 15±3, 17±3, 20±5, or 30±5.

In certain embodiments, the R³ moiety of the R¹ group of formula I is—N₃.

In other embodiments, the R³ moiety of the R¹ group of formula I is—CH₃.

In some embodiments, the R³ moiety of the R¹ group of formula I ishydrogen.

In certain embodiments, the R³ moiety of the R¹ group of formula I is anoptionally substituted aliphatic group. Examples include methyl,t-butyl, 5-norbornene-2-yl, octane-5-yl, acetylenyl,trimethylsilylacetylenyl, triisopropylsilylacetylenyl, andt-butyldimethylsilylacetylenyl. In some embodiments, said R³ moiety isan optionally substituted alkyl group. In other embodiments, said R³moiety is an optionally substituted alkynyl or alkenyl group. When saidR³ moiety is a substituted aliphatic group, substituents on R³ includeCN, N₃, trimethylsilyl, triisopropylsilyl, t-butyldimethylsilyl,N-methyl propiolamido, N-methyl-4-acetylenylanilino,N-methyl-4-acetylenylbenzoamido, bis-(4-ethynyl-benzyl)-amino,dipropargylamino, di-hex-5-ynyl-amino, di-pent-4-ynyl-amino,di-but-3-ynyl-amino, propargyloxy, hex-5-ynyloxy, pent-4-ynyloxy,di-but-3-ynyloxy, N-methyl-propargylamino, N-methyl-hex-5-ynyl-amino,N-methyl-pent-4-ynyl-amino, N-methyl-but-3-ynyl-amino,2-hex-5-ynyldisulfanyl, 2-pent-4-ynyldisulfanyl, 2-but-3-ynyldisulfanyl,and 2-propargyldisulfanyl. In certain embodiments, the R¹ group is2-(N-methyl-N-(ethynylcarbonyl)amino)ethoxy, 4-ethynylbenzyloxy, or2-(4-ethynylphenoxy)ethoxy.

In certain embodiments, the R³ moiety of the R¹ group of formula I is anoptionally substituted aryl group. Examples include optionallysubstituted phenyl and optionally substituted pyridyl. When said R³moiety is a substituted aryl group, substituents on R³ include CN, N₃,NO₂, —CH₃, —CH₂N₃, —CH═CH₂, —C≡CH, Br, I, F,bis-(4-ethynyl-benzyl)-amino, dipropargylamino, di-hex-5-ynyl-amino,di-pent-4-ynyl-amino, di-but-3-ynyl-amino, propargyloxy, hex-5-ynyloxy,pent-4-ynyloxy, di-but-3-ynyloxy, 2-hex-5-ynyloxy-ethyldisulfanyl,2-pent-4-ynyloxy-ethyldisulfanyl, 2-but-3-ynyloxy-ethyldisulfanyl,2-propargyloxy-ethyldisulfanyl, bis-benzyloxy-methyl,[1,3]dioxolan-2-yl, and [1,3]dioxan-2-yl.

In other embodiments, the R³ moiety of the R¹ group of formula I is aprotected aldehyde group. In certain embodiments the protected aldehydomoiety of R³ is an acyclic acetal, a cyclic acetal, a hydrazone, or animine. Exemplary R³ groups include dimethyl acetal, diethyl acetal,diisopropyl acetal, dibenzyl acetal, bis(2-nitrobenzyl) acetal,1,3-dioxane, 1,3-dioxolane, and semicarbazone. In certain embodiments,R³ is an acyclic acetal or a cyclic acetal. In other embodiments, R³ isa dibenzyl acetal.

In yet other embodiments, the R³ moiety of the R¹ group of formula I isa protected carboxylic acid group. In certain embodiments, the protectedcarboxylic acid moiety of R³ is an optionally substituted ester selectedfrom C₁₋₆ aliphatic or aryl, or a silyl ester, an activated ester, anamide, or a hydrazide. Examples of such ester groups include methyl,ethyl, propyl, isopropyl, butyl, isobutyl, benzyl, and phenyl ester. Inother embodiments, the protected carboxylic acid moiety of R³ is anoxazoline or an ortho ester. Examples of such protected carboxylic acidmoieties include oxazolin-2-yl and 2-methoxy-[1,3]dioxin-2-yl. Incertain embodiments, the R¹ group is oxazolin-2-ylmethoxy or2-oxazolin-2-yl-1-propoxy.

In still other embodiments, the R³ moiety of the R¹ group of formula Iis a detectable moiety. According to one aspect of the invention, the R³moiety of the R¹ group of formula I is a fluorescent moiety. Suchfluorescent moieties are well known in the art and include coumarins,quinolones, benzoisoquinolones, hostasol, and Rhodamine dyes, to namebut a few. Exemplary fluorescent moieties of the R³ group of R¹ includeanthracen-9-yl, pyren-4-yl, 9-H-carbazol-9-yl, the carboxylate ofrhodamine B, and the carboxylate of coumarin 343. In certainembodiments, the R³ moiety of the R¹ group of formula I is a detectablemoiety selected from:

In certain embodiments, the R³ moiety of the R¹ group of formula I is agroup suitable for Click chemistry. Click reactions tend to involvehigh-energy (“spring-loaded”) reagents with well-defined reactioncoordinates, giving rise to selective bond-forming events of wide scope.Examples include the nucleophilic trapping of strained-ringelectrophiles (epoxide, aziridines, aziridinium ions, episulfoniumions), certain forms of carbonyl reactivity (aldehydes and hydrazines orhydroxylamines, for example), and several types of cycloadditionreactions. The azide-alkyne 1,3-dipolar cycloaddition is one suchreaction. Click chemistry is known in the art and one of ordinary skillin the art would recognize that certain R³ moieties of the presentinvention are suitable for Click chemistry.

Compounds of formula I having R³ moieties suitable for Click chemistryare useful for conjugating said compounds to biological systems ormacromolecules such as proteins, viruses, and cells, to name but a few.The Click reaction is known to proceed quickly and selectively underphysiological conditions. In contrast, most conjugation reactions arecarried out using the primary amine functionality on proteins (e.g.lysine or protein end-group). Because most proteins contain a multitudeof lysines and arginines, such conjugation occurs uncontrollably atmultiple sites on the protein. This is particularly problematic whenlysines or arginines are located around the active site of an enzyme orother biomolecule. Thus, another embodiment of the present inventionprovides a method of conjugating the R¹ groups of a compound of formulaI to a macromolecule via Click chemistry. Yet another embodiment of thepresent invention provides a macromolecule conjugated to a compound offormula I via the R¹ group.

According to one embodiment, the R³ moiety of the R¹ group of formula Iis an azide-containing group. According to another embodiment, the R³moiety of the R¹ group of formula I is an alkyne-containing group. Incertain embodiments, the R³ moiety of the R¹ group of formula I has aterminal alkyne moiety. In other embodiments, R³ moiety of the R¹ groupof formula I is an alkyne moiety having an electron withdrawing group.Accordingly, in such embodiments, the R³ moiety of the R¹ group offormula I is

wherein E is an electron withdrawing group and y is 0-6. Such electronwithdrawing groups are known to one of ordinary skill in the art. Incertain embodiments, E is an ester. In other embodiments, the R³ moietyof the R¹ group of formula I is

wherein E is an electron withdrawing group, such as a —C(O)O— group andy is 0-6.

Certain metal-free click moieties are known in the literature. Examplesinclude 4-dibenzocyclooctynol (DIBO)

(from Ning et. al; Angew Chem Int Ed, 2008, 47, 2253); difluorinatedcyclooctynes (DIFO or DFO)

(from Codelli, et. al.; J. Am. Chem. Soc. 2008, 130, 11486-11493.);biarylazacyclooctynone (BARAC)

(from Jewett et. al.; J. Am. Chem. Soc. 2010, 132, 3688.); orbicyclononyne (BCN)

(From Dommerholt, et. al.; Angew Chem Int Ed, 2010, 49, 9422-9425). Thepreparation of metal free click PEG derivatives is described in U.S.application Ser. No. 13/601,606, the entire contents of which are herebyincorporated by reference.

According to one embodiment, the R³ moiety of the R¹ group of formula Iis metal free click moiety. In another embodiment, the R³ moiety of theR¹ group of formula I is an optionally substituted strained cyclooctynemoiety. In certain embodiments, the R³ moiety of the R¹ group of formulaI is a metal free click moiety selected from:

As defined generally above, Q is a valence bond or a bivalent, saturatedor unsaturated, straight or branched C₁₋₁₂ hydrocarbon chain, wherein0-6 methylene units of Q are independently replaced by -Cy-, —O—, —NH—,—S—, —OC(O)—, —C(O)O—, —C(O)—, —SO—, —SO₂—, —NHSO₂—, —SO₂NH—, —NHC(O)—,—C(O)NH—, —OC(O)NH—, or —NHC(O)O—, wherein -Cy- is an optionallysubstituted 5-8 membered bivalent, saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, or an optionally substituted 8-10 membered bivalentsaturated, partially unsaturated, or aryl bicyclic ring having 0-5heteroatoms independently selected from nitrogen, oxygen, or sulfur. Incertain embodiments, Q is a valence bond. In other embodiments, Q is abivalent, saturated C₁₋₁₂ alkylene chain, wherein 0-6 methylene units ofQ are independently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—,or —C(O)—, wherein -Cy- is an optionally substituted 5-8 memberedbivalent, saturated, partially unsaturated, or aryl ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, oran optionally substituted 8-10 membered bivalent saturated, partiallyunsaturated, or aryl bicyclic ring having 0-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

In certain embodiments, Q is -Cy- (i.e. a C₁ alkylene chain wherein themethylene unit is replaced by -Cy-), wherein -Cy- is an optionallysubstituted 5-8 membered bivalent, saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. According to one aspect of the present invention,-Cy- is an optionally substituted bivalent aryl group. According toanother aspect of the present invention, -Cy- is an optionallysubstituted bivalent phenyl group. In other embodiments, -Cy- is anoptionally substituted 5-8 membered bivalent, saturated carbocyclicring. In still other embodiments, -Cy- is an optionally substituted 5-8membered bivalent, saturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. Exemplary -Cy-groups include bivalent rings selected from phenyl, pyridyl,pyrimidinyl, cyclohexyl, cyclopentyl, or cyclopropyl.

As defined above, R^(x) is a hydoxamate or catechol containing moiety.In certain embodiments, R^(x) is a hydroxamic acid containing moiety. Inother embodiments, R^(x) is a catechol containing moiety. In certainembodiments, R^(x) is selected from

In certain embodiments, R^(x) is selected from:

As defined above, R^(y) is selected from one or more natural orunnatural amino acid side chain groups such that the overall block ishydrophobic. Such hydrophobic amino acid side-chain groups include anoptionally protected tyrosine side-chain, an optionally protected serineside-chain, an optionally protected threonine side-chain, phenylalanine,alanine, valine, leucine, tryptophan, proline, benzyl and alkylglutamates, or benzyl and alkyl aspartates or mixtures thereof. One ofordinary skill in the art would recognize that protection of a polar orhydrophilic amino acid side-chain can render that amino acid nonpolar.For example, a suitably protected tyrosine hydroxyl group can renderthat tyrosine nonpolar and hydrophobic by virtue of protecting thehydroxyl group. Protecting groups for the hydroxyl, amino, and thiol,and carboylate functional groups of R^(y) are as described herein.Furthermore, one of ordinary skill in the art would recognize thathydrophilic and hydrophobic amino acid side chains can be combined suchthat the overall block is hydrophobic. For example, a majority ofleucine side chain groups can be combined with a minority of asparticacid side chain groups wherein the resulting block is net hydrophobic.Such mixtures of amino acid side-chain groups include tyrosine andleucine, tyrosine and phenylalanine, serine and phenylalanine, asparticacid and phenylalanine, glutamic acid and phenylalanine, tyrosine andbenzyl glutamate, serine and benzyl glutamate, aspartic acid and benzylglutamate, glutamic acid and benzyl glutamate, aspartic acid andleucine, and glutamic acid and leucine.

In some embodiments, Ry consists of a mixture of three natural orunnatural amino acid side chain groups such that the overall block ishydrophobic. Such ternary mixtures of amino acid side-chain groupsinclude, but are not limited to: leucine, tyrosine, and aspartic acid;leucine, tyrosine, and glutamic acid; phenylalanine, tyrosine, andaspartic acid; or phenylalanine, tyrosine, and glutamic acid.

In other embodiments, R^(y) consists of a mixture of D-hydrophobic andL-hydrophilic amino acid side-chain groups such that the overallpoly(amino acid) block comprising R^(y) is hydrophobic and is a mixtureof D- and L-configured amino acids. Such mixtures of amino acidside-chain groups include L-tyrosine and D-leucine, L-tyrosine andD-phenylalanine, L-serine and D-phenylalanine, L-aspartic acid andD-phenylalanine, L-glutamic acid and D-phenylalanine, L-tyrosine andD-benzyl glutamate, L-serine and D-benzyl glutamate, L-aspartic acid andD-benzyl glutamate, L-glutamic acid and D-benzyl glutamate, L-asparticacid and D-leucine, and L-glutamic acid and D-leucine. Ratios(D-hydrophobic to L-hydrophilic) of such mixtures include any of 6:1,5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4; 1:5, and 1:6.

As defined generally above, the R² group of formula I is amono-protected amine, a di-protected amine, —NHR⁴, —N(R⁴)₂, —NHC(O)R⁴,—NR⁴C(O)R⁴, —NR⁴C(O)NHR⁴, —NHC(O)N(R⁴)₂, —NR⁴C(O)NHR⁴, —NR⁴C(O)N(R⁴)₂,—NHC(O)OR⁴, —NR⁴C(O)OR⁴, —NHSO₂R⁴, or —NR⁴SO₂R⁴, wherein each R⁴ isindependently an optionally substituted group selected from aliphatic, a5-8 membered saturated, partially unsaturated, or aryl ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, an8-10-membered saturated, partially unsaturated, or aryl bicyclic ringhaving 0-5 heteroatoms independently selected from nitrogen, oxygen, orsulfur, or a detectable moiety, or two R⁴ on the same nitrogen atom aretaken together with said nitrogen atom to form an optionally substituted4-7 membered saturated, partially unsaturated, or aryl ring having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, the R² group of formula I is —NHR⁴ or —N(R⁴)₂wherein each R⁴ is an optionally substituted aliphatic group. Oneexemplary R⁴ group is 5-norbornen-2-yl-methyl. According to yet anotheraspect of the present invention, the R^(2a) group of formula I is —NHR⁴wherein R⁴ is a C₁₋₆ aliphatic group substituted with N₃. Examplesinclude —CH₂N₃. In some embodiments, R⁴ is an optionally substitutedC₁₋₆ alkyl group. Examples include methyl, ethyl, propyl, butyl, pentyl,hexyl, 2-(tetrahydropyran-2-yloxy)ethyl, pyridin-2-yldisulfanylmethyl,methyldisulfanylmethyl, (4-acetylenylphenyl)methyl,3-(methoxycarbonyl)-prop-2-ynyl, methoxycarbonylmethyl,2-(N-methyl-N-(4-acetylenylphenyl)carbonylamino)-ethyl,2-phthalimidoethyl, 4-bromobenzyl, 4-chlorobenzyl, 4-fluorobenzyl,4-iodobenzyl, 4-propargyloxybenzyl, 2-nitrobenzyl,4-(bis-4-acetylenylbenzyl)aminomethyl-benzyl, 4-propargyloxy-benzyl,4-dipropargylamino-benzyl, 4-(2-propargyloxy-ethyldisulfanyl)benzyl,2-propargyloxy-ethyl, 2-propargyldisulfanyl-ethyl, 4-propargyloxy-butyl,2-(N-methyl-N-propargylamino)ethyl, and2-(2-dipropargylaminoethoxy)-ethyl. In other embodiments, R⁴ is anoptionally substituted C₂₋₆ alkenyl group. Examples include vinyl,allyl, crotyl, 2-propenyl, and but-3-enyl. When R⁴ group is asubstituted aliphatic group, substituents on R⁴ include N₃, CN, andhalogen. In certain embodiments, R⁴ is —CH₂CN, —CH₂CH₂CN, —CH₂CH(OCH₃)₂,4-(bisbenzyloxymethyl)phenylmethyl, and the like.

According to another aspect of the present invention, the R² group offormula I is —NHR⁴ wherein R⁴ is an optionally substituted C₂ alkynylgroup. Examples include —CC≡CH, —CH₂C≡CH, —CH₂C≡CCH₃, and —CH₂CH₂C≡CH.

In certain embodiments, the R² group of formula I is —NHR⁴ wherein R⁴ isan optionally substituted 5-8-membered aryl ring. In certainembodiments, R⁴ is optionally substituted phenyl or optionallysubstituted pyridyl. Examples include phenyl,4-t-butoxycarbonylaminophenyl, 4-azidomethylphenyl,4-propargyloxyphenyl, 2-pyridyl, 3-pyridyl, and 4-pyridyl. In certainembodiments, R^(2a) is 4-t-butoxycarbonylaminophenylamino,4-azidomethylphenamino, or 4-propargyloxyphenylamino.

In certain embodiments, the R^(2a) group of formula I is —NHR⁴ whereinR⁴ is an optionally substituted phenyl ring. Substituents on the R⁴phenyl ring include halogen; —(CH₂)₀₋₄R^(∘); —(CH₂)₀₋₄OR^(∘);—(CH₂)₀₋₄CH(OR^(∘))₂; —(CH₂)₀₋₄SR^(∘); —(CH₂)₀₋₄Ph, which may besubstituted with R^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(∘); —CH═CHPh, which may be substituted with R^(∘); —NO₂; —CN;—N₃; —(CH₂)₀₋₄N(R^(∘))₂; —(CH₂)₀₋₄N(R^(∘))C(O)R^(∘); —N(R^(∘))C(S)R^(∘);—(CH₂)₀₋₄N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))C(S)NR^(∘) ₂;—(CH₂)₀₋₄N(R^(∘))C(O)OR^(∘); —N(R^(∘))N(R^(∘))C(O)R^(∘);—N(R^(∘))N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))N(R^(∘))C(O)OR^(∘);—(CH₂)₀₋₄C(O)R^(∘); —C(S)R^(∘); —(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄C(O)SR^(∘); —(CH₂)₀₋₄C(O)OSiR^(∘) ₃; —(CH₂)₀₋₄OC(O)R^(∘);—(CH₂)₀₋₄SC(O)R^(∘); —(CH₂)₀₋₄C(O)NR^(∘) ₂; —C(S)NR^(∘) ₂;—CH₂)₀₋₄OC(O)NR^(∘) ₂; —C(O)N(OR^(∘))R^(∘); —C(O)C(O)R^(∘);—C(O)CH₂C(O)R^(∘); —C(NOR^(∘))R^(∘); —(CH₂)₀₋₄SSR^(∘);—(CH₂)₀₋₄S(O)₂R^(∘); —(CH₂)₀₋₄S(O)₂OR^(∘); —(CH₂)₀₋₄OS(O)₂R^(∘);—S(O)₂NR^(∘) ₂; —(CH₂)₀₋₄S(O)R^(∘); —N(R^(∘))S(O)₂NR^(∘) ₂;—N(R^(∘))S(O)₂R^(∘); —N(OR^(∘))R^(∘); —C(NH)NR^(∘) ₂; —P(O)₂R^(∘);—P(O)R^(∘) ₂; —OP(O)R^(∘) ₂; SiR^(∘) ₃; wherein each independentoccurrence of R^(∘) is as defined herein supra. In other embodiments,the R^(2a) group of formula I is —NHR⁴ wherein R⁴ is phenyl substitutedwith one or more optionally substituted C₁₋₆ aliphatic groups. In stillother embodiments, R⁴ is phenyl substituted with vinyl, allyl,acetylenyl, —CH₂N₃, —CH₂CH₂N₃, —CH₂C≡CCH₃, or —CH₂C≡CH.

In certain embodiments, the R² group of formula I is —NHR⁴ wherein R⁴ isphenyl substituted with N₃, N(R^(∘))₂, CO₂R^(∘), or C(O)R^(∘) whereineach R^(∘) is independently as defined herein supra.

In certain embodiments, the R² group of formula I is —N(R⁴)₂ whereineach R⁴ is independently an optionally substituted group selected fromaliphatic, phenyl, naphthyl, a 5-6 membered aryl ring having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, ora 8-10 membered bicyclic aryl ring having 1-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, or a detectable moiety.

In other embodiments, the R² group of formula I is —N(R⁴)₂ wherein thetwo R⁴ groups are taken together with said nitrogen atom to form anoptionally substituted 4-7 membered saturated, partially unsaturated, oraryl ring having 1-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. According to another embodiment, the two R⁴ groupsare taken together to form a 5-6-membered saturated or partiallyunsaturated ring having one nitrogen wherein said ring is substitutedwith one or two oxo groups. Such R^(2a) groups include, but are notlimited to, phthalimide, maleimide and succinimide.

In certain embodiments, the R² group of formula I is a mono-protected ordi-protected amino group. In certain embodiments R^(2a) is amono-protected amine. In certain embodiments R^(2a) is a mono-protectedamine selected from aralkylamines, carbamates, allyl amines, or amides.Exemplary mono-protected amino moieties include t-butyloxycarbonylamino,ethyloxycarbonylamino, methyloxycarbonylamino,trichloroethyloxy-carbonylamino, allyloxycarbonylamino,benzyloxocarbonylamino, allylamino, benzylamino,fluorenylmethylcarbonyl, formamido, acetamido, chloroacetamido,dichloroacetamido, trichloroacetamido, phenylacetamido,trifluoroacetamido, benzamido, and t-butyldiphenylsilylamino. In otherembodiments R^(2a) is a di-protected amine. Exemplary di-protected aminomoieties include di-benzylamino, di-allylamino, phthalimide, maleimido,succinimido, pyrrolo, 2,2,5,5-tetramethyl-[1,2,5]azadisilolidino, andazido. In certain embodiments, the R^(2a) moiety is phthalimido. Inother embodiments, the R^(2a) moiety is mono- or di-benzylamino or mono-or di-allylamino.

In certain embodiments, the present invention provides a triblockcopolymer of formula II:

wherein:

-   -   n is 20-500;    -   m is 0, 1, or 2;    -   x is 3 to 50;    -   y is 5 to 100;    -   R^(y) is selected from one or more natural or unnatural amino        acid side chain groups such that the overall block is        hydrophobic;    -   R¹ is —Z(CH₂CH₂Y)_(p)(CH₂)_(t)R³, wherein:        -   Z is —O—, —NH—, —S—, —C≡C—, or —CH₂—;        -   each Y is independently —O— or —S—;        -   p is 0-10;        -   t is 0-10; and    -   R³ is hydrogen, —N₃, —CN, —NH₂, —CH₃,

-   -    a strained cyclooctyne moiety, a mono-protected amine, a        di-protected amine, an optionally protected aldehyde, an        optionally protected hydroxyl, an optionally protected        carboxylic acid, an optionally protected thiol, or an optionally        substituted group selected from aliphatic, a 5-8 membered        saturated, partially unsaturated, or aryl ring having 0-4        heteroatoms independently selected from nitrogen, oxygen, or        sulfur, an 8-10 membered saturated, partially unsaturated, or        aryl bicyclic ring having 0-5 heteroatoms independently selected        from nitrogen, oxygen, or sulfur, or a detectable moiety.

In certain embodiments, a triblock copolymer of Formula II is selectedfrom the following exemplary compounds shown in Table 1,

wherein n is 20 to 500, x is 3 to 50, y′ is 3 to 50, and y″ is 3 to 50.

TABLE 1 Compound # R¹ R^(ya) R^(yb) 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

In certain embodiments, a triblock copolymer of Formula II is

In certain embodiments, a triblock copolymer of Formula II is

In certain embodiments, a triblock copolymer of Formula II is

In certain embodiments, the present invention provides a triblockcopolymer of formula III:

-   -   wherein:        -   n is 20-500;        -   m is 0, 1, or 2;        -   x is 3 to 50;        -   y is 5 to 100;        -   R^(y) is selected from one or more natural or unnatural            amino acid side chain groups such that the overall block is            hydrophobic;        -   R¹ is —Z(CH₂CH₂Y)_(p)(CH₂)_(t)R³, wherein:            -   Z is —O—, —NH—, —S—, —C≡C—, or —CH₂—;            -   each Y is independently —O— or —S—;            -   p is 0-10;            -   t is 0-10; and        -   R³ is hydrogen, —N₃, —CN, —NH₂, —CH₃,

-   -   -    a strained cyclooctyne moiety, a mono-protected amine, a            di-protected amine, an optionally protected aldehyde, an            optionally protected hydroxyl, an optionally protected            carboxylic acid, an optionally protected thiol, or an            optionally substituted group selected from aliphatic, a 5-8            membered saturated, partially unsaturated, or aryl ring            having 0-4 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, an 8-10 membered saturated, partially            unsaturated, or aryl bicyclic ring having 0-5 heteroatoms            independently selected from nitrogen, oxygen, or sulfur, or            a detectable moiety.

In certain embodiments, a triblock copolymer of Formula III is selectedfrom the following exemplary compounds shown in Table 2,

wherein n is 20 to 500, x is 3 to 50, y′ is 3 to 50, and y is 3 to 50.

TABLE 2 81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

In certain embodiments, the present invention provides a triblockcopolymer of formula IV:

-   -   wherein:        -   n is 20-500;        -   m is 0, 1, or 2;        -   x is 3 to 50;        -   y is 5 to 100;        -   R^(y) is selected from one or more natural or unnatural            amino acid side chain groups such that the overall block is            hydrophobic;        -   R¹ is —Z(CH₂CH₂Y)_(p)(CH₂)_(t)R³, wherein:            -   Z is —O—, —NH—, —S—, —C≡C—, or —CH₂—;            -   each Y is independently —O— or —S—;            -   p is 0-10;            -   t is 0-10; and        -   R³ is hydrogen, —N₃, —CN, —NH₂, —CH₃,

-   -   -    a strained cyclooctyne moiety, a mono-protected amine, a            di-protected amine, an optionally protected aldehyde, an            optionally protected hydroxyl, an optionally protected            carboxylic acid, an optionally protected thiol, or an            optionally substituted group selected from aliphatic, a 5-8            membered saturated, partially unsaturated, or aryl ring            having 0-4 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, an 8-10 membered saturated, partially            unsaturated, or aryl bicyclic ring having 0-5 heteroatoms            independently selected from nitrogen, oxygen, or sulfur, or            a detectable moiety.

B. Targeting Group Attachment

Compounds of any of formulae I, II, III, and IV having R³ moietiessuitable for Click chemistry are useful for conjugating said compoundsto biological systems or macromolecules such as peptides, proteins,viruses, and cells, to name but a few. The Click reaction is known toproceed quickly and selectively under physiological conditions. Incontrast, most conjugation reactions are carried out using the primaryamine functionality on proteins (e.g. lysine or protein end-group).Because most proteins contain a multitude of lysines and arginines, suchconjugation occurs uncontrollably at multiple sites on the protein. Thisis particularly problematic when lysines or arginines are located aroundthe active site of an enzyme or other biomolecule. Thus, anotherembodiment of the present invention provides a method of conjugating theR¹ groups of a compound of any of formulae I, II, III, and IV to amacromolecule via Click chemistry. Yet another embodiment of the presentinvention provides a macromolecule conjugated to a compound of any ofany of formulae I, II, III, and IV via the R¹ group.

After incorporating the poly (amino acid) block portions into themulti-block coploymer of the present invention resulting in amulti-block copolymer of the form W—X—X′, the other end-groupfunctionality, corresponding to the R¹ moiety of any of formulae I, II,III, and IV can be used to attach targeting groups for cell specificdelivery including, but not limited to, attach targeting groups for cellspecific delivery including, but not limited to, proteins,oliogopeptides, antibodies, monosaccarides, oligosaccharides, vitamins,or other small biomolecules. Such targeting groups include, but are notlimited to monoclonal and polyclonal antibodies (e.g. IgG, IgA, IgM,IgD, IgE antibodies), sugars (e.g. mannose, mannose-6-phosphate,galactose), proteins (e.g. Transferrin), oligopeptides (e.g. cyclic andacylic RGD-containing oligopedptides), and vitamins (e.g. folate).Alternatively, the R¹ moiety of any of formulae I, II, III, and IV isbonded to a biomolecule, drug, cell, or other substrate.

In other embodiments, the R¹ moiety of any of formulae I, II, III, andIV is bonded to biomolecules which promote cell entry and/or endosomalescape. Such biomolecules include, but are not limited to, oligopeptidescontaining protein transduction domains such as the HIV Tat peptidesequence (GRKKRRQRRR) or oligoarginine (RRRRRRRRR). Oligopeptides whichundergo conformational changes in varying pH environments sucholigohistidine (HHHHH) also promote cell entry and endosomal escape.

In other embodiments, the R¹ moiety of any of formulae I, II, III, andIV is bonded to detectable moieties, such as fluorescent dyes or labelsfor positron emission tomography including molecules containingradioisotopes (e.g. ¹⁸F) or ligands with bound radioactive metals (e.g.⁶²Cu). In other embodiments, the R¹ moiety of any of formulae I, II,III, and IV is bonded to a contrast agents for magnetic resonanceimaging such as gadolinium, gadolinium chelates, or iron oxide (e.gFe₃O₄ and Fe₂O₃) particles. In other embodiments, the R¹ moiety of anyof formulae I, II, III, and IV is bonded to a semiconductingnanoparticle such as cadmium selenide, cadmium sulfide, or cadmiumtelluride or bonded to other metal nanoparticles such as colloidal gold.In other embodiments, the R¹ moiety of any of formulae I, II, III, andIV is bonded to natural or synthetic surfaces, cells, viruses, dyes,drugs, chelating agents, or used for incorporation into hydrogels orother tissue scaffolds.

In one embodiment, the R¹ moiety of any of formulae I, II, III, and IVis an alkyne or a terminal alkyne derivative which is capable ofundergoing [3+2] cycloaddition reactions with complementaryazide-bearing molecules and biomolecules. In another embodiment, the R¹moiety of any of formulae I, II, III, and IV is an azide or an azidederivative which is capable of undergoing [3+2] cycloaddition reactionswith complementary alkyne-bearing molecules and biomolecules (i.e. clickchemistry).

Click chemistry has become a popular method of bioconjugation due to itshigh reactivity and selectivity, even in biological media. See Kolb, H.C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int. Ed. 2001, 40,2004-2021; and Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.;Sharpless, K. B.; Finn, M. G. J. Am. Chem. Soc. 2003, 125, 3192-3193. Inaddition, currently available recombinant techniques permit theintroduction of azides and alkyne-bearing non-canonical amino acids intoproteins, cells, viruses, bacteria, and other biological entities thatconsist of or display proteins. See Link, A. J.; Vink, M. K. S.;Tirrell, D. A. J. Am. Chem. Soc. 2004, 126, 10598-10602; Deiters, A.;Cropp, T. A.; Mukherji, M.; Chin, J. W.; Anderson, C.; Schultz, P. G. J.Am. Chem. Soc. 2003, 125, 11782-11783.

In another embodiment, the [3+2] cycloaddition reaction of azide oracetylene-bearing nanovectors and complimentary azide oracetylene-bearing biomolecules are transition metal catalyzed.Copper-containing molecules which catalyze the “click” reaction include,but are not limited to, copper bromide (CuBr), copper chloride (CuCl),copper sulfate (CuSO₄), copper iodide (CuI), [Cu(MeCN)₄](OTf), and[Cu(MeCN)₄](PF₆). Organic and inorganic metal-binding ligands can beused in conjunction with metal catalysts and include, but are notlimited to, sodium ascorbate, tris(triazolyl)amine ligands,tris(carboxyethyl)phosphine (TCEP), and sulfonated bathophenanthrolineligands.

In another embodiment, the R¹ moiety of any of formulae I, II, III, andIV is an hydrazine or hydrazide derivative which is capable ofundergoing reaction with biomolecules containing aldehydes or ketones toform hydrazone linkages. In another embodiment, the R¹ moiety of any offormulae I, II, III, and IV is an aldehyde or ketone derivative which iscapable of undergoing reaction with biomolecules containing a hydrazineor hydrazide derivative to form hydrazone linkages.

In another embodiment, the R¹ moiety of any of formulae I, II, III, andIV is a hydroxylamine derivative which is capable of undergoing reactionwith biomolecules containing aldehydes or ketones. In anotherembodiment, the R¹ moiety of any of formulae I, II, III, and IV is analdehyde or ketone which is capable of undergoing reaction withbiomolecules containing a hydroxylamine, or a hydroxylamine derivative.

In yet another embodiment, the R¹ moiety of any of formulae I, II, III,and IV is an aldehyde or ketone derivative which is capable ofundergoing reaction with biomolecules containing primary or secondaryamines to form imine linkages. In another embodiment, the R¹ moiety ofany of formulae I, II, III, and IV is a primary or secondary amine whichis capable of undergoing reaction with biomolecules containing analdehyde or ketone functionality to form imine linkages. It will beappreciated that imine linkages can be further converted to stable aminelinkages by treatment with a reducing agent (e.g. lithium aluminumhydride, sodium borohydride, sodium cyanoborohydride, etc.)

In yet another embodiment, the R¹ moiety of any of formulae I, II, III,and IV is an amine (primary or secondary) or alcohol which is capable ofundergoing reaction with biomolecules containing activated esters (e.g.4-nitrophenol ester, N-hydroxysuccinimide, pentafluorophenyl ester,ortho-pyridylthioester), to form amide or ester linkages. In still otherembodiments, the R¹ moiety of any of formulae I, II, III, and IV is anactivated ester which is capable of undergoing reaction withbiomolecules possessing amine (primary or secondary) or alcohols to formamide or ester linkages.

In still other embodiments, the R¹ moiety of any of formulae I, II, III,and IV is an amine or alcohol which is bound to biomolecules withcarboxylic acid functionality using a coupling agent. In still otherembodiments, the R¹ moiety of any of formulae I, II, III, and IV is acarboxylic acid functionality which is bound to biomolecules containingamine or alcohol functionality using a coupling agent. Such couplingagents include, but are not limited to, carbodiimides (e.g.1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), diisopropylcarbodiimide (DIC), dicyclohexyl carbodiimide (DCC)), aminium orphosphonium derivatives (e.g. PyBOP, PyAOP, TBTU, HATU, HBTU), or acombination of 1-hydroxybenzotriazole (HOBt) and a aminium orphosphonium derivative.

In another embodiment, the R¹ moiety of any of formulae I, II, III, andIV is an electrophile such as maleimide, a maleimide derivative, or abromoacetamide derivative, which is capable of reaction withbiomolecules containing thiols or amines. In another embodiment, the R¹moiety of any of formulae I, II, III, and IV is a nucleophile such as anamine or thiol which is capable or reaction with biomolecules containingelectrophilic functionality such as maleimide, a maleimide derivative,or a bromoacetamide derivative.

In still other embodiments, the R¹ moiety of any of formulae I, II, III,and IV is a ortho-pyridyl disulfide moiety which undergoes disulfideexchange with biomolecules containing thiol functionality. In stillother embodiments, the R¹ moiety of any of formulae I, II, III, and IVis a thiol or thiol derivative which undergoes disulfide exchange withbiomolecules containing ortho-pyridyl disulfide functionality. It willbe appreciated that such exchange reactions result in a disulfidelinkage, which is reversible in the presence of a reducing agent (e.g.glutathione, dithiothreitol (DTT), etc.).

In certain embodiments, micelles of the present invention are mixedmicelles comprising one or more compounds of formulae I, II, III, andIV. It will be appreciated that mixed micelles having different R¹groups, as described herein, can be conjugated to multiple othercompounds and/or macromolecules. For example, a mixed micelle of thepresent invention can have one R¹ group suitable for Click chemistry andanother R¹ group suitable for covalent attachment via a variety ofcoupling reactions. Such a mixed micelle can be conjugated to differentcompounds and/or macromolecules via these different R¹ groups. Suchconjugation reactions are well known to one of ordinary skill in the artand include those described herein.

In certain embodiments, the present invention provides a triblockcopolymer of formula V:

-   -   wherein each of Q, x, y, n, R^(x), R^(y) and R² is as defined        above and as described in classes and subclasses herein, both        singly and in combination;        -   J is independently a valence bond or a bivalent, saturated            or unsaturated, straight or branched C₁₋₁₂ hydrocarbon            chain, wherein 0-6 methylene units of Q are independently            replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—, —C(O)—,            —SO—, —SO₂—, —NHSO₂—, —SO₂NH—, —NHC(O)—, —C(O)NH—,            —OC(O)NH—, or —NHC(O)O—, wherein:            -   -Cy- is an optionally substituted 5-8 membered bivalent,                saturated, partially unsaturated, or aryl ring having                0-4 heteroatoms independently selected from nitrogen,                oxygen, or sulfur, or an optionally substituted 8-10                membered bivalent saturated, partially unsaturated, or                aryl bicyclic ring having 0-5 heteroatoms independently                selected from nitrogen, oxygen, or sulfur;    -   each T is independently a targeting group.

As generally described above, T is a targeting group. Such targetinggroups are described in detail in United States patent applicationpublication number 2009/0110662, published Apr. 30, 2009, the entiretyof which is hereby incorporated by reference. Additional targetinggroups are described in detail in U.S. patent application Ser. No.13/415,910, filed Mar. 9, 2012, the entirety of which is herebyincorporated by reference.

In certain embodiments, the J group is a valence bond as describedabove. In certain embodiments, the J group is a methylene group. Inother embodiments, the J group is a carbonyl group. In certainembodiments, the J group of Formula V is a valence bond. In otherembodiments, the J group is represented by a moiety in Table 3.

TABLE 3

a

b

c

d

e

f

g

h

i

C. Micelle Formation

Amphiphilic multiblock copolymers, as described herein, canself-assemble in aqueous solution to form nano- and micron-sizedstructures. In water, these amphiphilic multiblock copolymers assembleby multi-molecular micellization when present in solution above thecritical micelle concentration (CMC). Without wishing to be bound by anyparticular theory, it is believed that the hydrophobic poly(amino acid)portion or “block” of the copolymer collapses to form the micellar core,while the hydrophilic PEG block forms a peripheral corona and impartswater solubility. In certain embodiments, the multiblock copolymers inaccordance with the present invention possess distinct hydrophobic andhydrophilic segments that form micelles. In addition, these multiblockpolymers optionally comprise a poly(amino acid) block which containsfunctionality for crosslinking. It will be appreciated that thisfunctionality is found on the corresponding amino acid side-chain.

D. Drug Loading

According to one embodiment, the present invention provides a micellecomprising a triblock copolymer which comprises a polymeric hydrophilicblock, optionally a crosslinkable or crosslinked poly(amino acid block),and a hydrophobic D,L-mixed poly(amino acid) block, characterized inthat said micelle has an inner core, optionally a crosslinkable orcrosslinked outer core, and a hydrophilic shell. As described herein,micelles of the present invention are especially useful forencapsulating therapeutic agents. In certain embodiments the therapeuticagent is hydrophobic.

Without wishing to be bound by any particular theory, it is believedthat the accommodation of structurally diverse therapeutic agents withina micelle of the present invention is effected by adjusting thehydrophobic D,L-mixed poly(amino acid) block, i.e., the block comprisingR^(y). As discussed above, the hydrophobic mixture of D and Lstereoisomers affords a poly(amino acid) block with a random coilconformation thereby enhancing the encapsulation of hydrophobic drugs.

Hydrophobic small molecule drugs suitable for loading into micelles ofthe present invention are well known in the art. In certain embodiments,the present invention provides a drug-loaded micelle as describedherein. In other embodiments, the present invention provides adrug-loaded micelle as described herein, wherein the drug is ahydrophobic drug selected from those described herein, infra.

As used herein, the terms hydrophobic small molecule drugs, smallmolecule drugs, therapeutic agent, and hydrophobic therapeutic agentsare all interchangable.

According to another embodiment, the present invention provides adrug-loaded micelle comprising a triblock copolymer of formula I and atherapeutic agent.

According to another embodiment, the present invention provides adrug-loaded micelle comprising a triblock copolymer of formula I and ahydrophobic therapeutic agent.

In other embodiments, the present invention provides a system comprisinga triblock copolymer of formula I and a hydrophobic therapeutic agent.In another embodiment, the present invention provides a systemcomprising a triblock copolymer of any of formulae I, II, III, or IV,either singly or in combination, and a hydrophobic therapeutic agent. Inyet another embodiment, the present invention provides a systemcomprising a triblock copolymer of formula II and a hydrophobictherapeutic agent.

In some embodiments, the present invention provides a micelle, having asuitable hydrophobic therapeutic agent encapsulated therein, comprisinga multiblock copolymer of formula I and a multiblock copolymer offormula V, wherein each of formula I and formula V are as defined aboveand described herein, wherein the ratio of Formula I to Formula V isbetween 1000:1 and 1:1. In other embodiments, the ratio is 1000:1,100:1, 50:1, 33:1, 25:1, 20:1, 10:1, 5:1, or 4:1. In yet otherembodiments, the ratio is between 100:1 and 25:1.

In some embodiments, the present invention provides a micelle, having anhydrophobic therapeutic agent encapsulated therein, comprising amultiblock copolymer of formula II and a multiblock copolymer of formulaV, wherein each of formula II and formula V are as defined above anddescribed herein, wherein the ratio of Formula II to Formula V isbetween 1000:1 and 1:1. In other embodiments, the ratio is 1000:1,100:1, 50:1, 33:1, 25:1, 20:1, 10:1, 5:1, or 4:1. In yet otherembodiments, the ratio is between 100:1 and 25:1.

Embodiments with respect to each of the R¹, R^(2a), Q, R^(x), R^(y), n,m, and m′ groups of formula I, are as described in various classes andsubclasses, both singly and in combination, herein.

In certain embodiments, the present invention provides a drug-loadedmicelle, as described herein, wherein the drug is a taxane.

Taxanes are well known in the literature and are natural productsproduced by plants of the genus Taxus. The mechanism of action ismicrotubule stabilization, thus inhibiting mitosis. Many taxanes arepoorly soluble or nearly completely insoluble in water. Exemplaryepothilones are shown below.

Epothilones are a group of molecules that have been shown to bemicrotubule stabilizers, a mechanism similar to paclitaxel (Bollag D Met al. Cancer Res. 1995, 55, 2325-2333). Biochemical studiesdemonstrated that epothilones can displace paclitaxel from tubulin,suggesting that they compete for the same binding site (Kowalski R J,Giannakakou P, Hamel E. J Biol Chem. 1997, 272, 2534-2541). Oneadvantage of the epothilones is that they exert much greater cytotoxiceffect in PGP overexpressing cells compared to paclitaxel. Exemplaryepothilones are shown below.

In certain embodiments, the present invention provides a drug-loadedmicelle, as described herein, wherein the drug is paclitaxel.

In certain embodiments, the present invention provides a drug-loadedmicelle, as described herein, wherein the drug is docetaxel.

In certain embodiments, the present invention provides a drug-loadedmicelle, as described herein, wherein the drug is cabazitaxel.

In certain embodiments, the present invention provides a drug-loadedmicelle, as described herein, wherein the drug is an epothilone.

In certain embodiments, the present invention provides a drug-loadedmicelle, as described herein, wherein the drug is Epothilone B orEpothilone D.

In certain embodiments, the present invention provides a drug-loadedmicelle, as described herein, wherein the drug is Epothilone A orEpothilone C.

Vinca alkaloids are well known in the literature and are a set ofanti-mitotic agents. Vinca alkaloids include vinblastine, vincristine,vindesine, and vinorelbine, and act to prevent the formation ofmicrotubules. Exemplary vinca alkaloids are shown below.

In certain embodiments, the present invention provides a drug-loadedmicelle, as described herein, wherein the drug is a vinca alkaloid.

In certain embodiments, the present invention provides a drug-loadedmicelle, as described herein, wherein the drug is vinorelbine.

Berberine is well known in the literature and shown pharmaceuticaleffects in a range of applications including antibacterial and oncologyapplications. The anti-tumor activity of berberine and associatedderivatives are described in Hoshi, et. al. Gann, 1976, 67, 321-325.Specifically, berberrubine and ester derivatives of berberrubine areshown to have increased anti-tumor activity with respect to berberine.The structures of berberine and berberrubine are shown below.

In certain embodiments, the present invention provides a drug-loadedmicelle, as described herein, wherein the drug is berberine.

In certain embodiments, the present invention provides a drug-loadedmicelle, as described herein, wherein the drug is berberrubine.

The antitumor plant alkaloid camptothecin (CPT) is a broad-spectrumanticancer agent that targets DNA topoisomerase I. Although CPT hasshown promising antitumor activity in vitro and in vivo, it has not beenclinically used because of its low therapeutic efficacy and severetoxicity. Among CPT analogues, irinotecan hydrochloride (CPT-11) hasrecently been shown to be active against colorectal, lung, and ovariancancer. CPT-11 itself is a prodrug and is converted to7-ethyl-10-hydroxy-CPT (known as SN-38), a biologically activemetabolite of CPT-11, by carboxylesterases in vivo. A number ofcamptothecin derivatives are in development, the structures of which areshown below.

In certain embodiments, the present invention provides a drug-loadedmicelle, as described herein, wherein the drug is a camptothecin.

In certain embodiments, the present invention provides a drug-loadedmicelle, as described herein, wherein the drug is SN-38.

In certain embodiments, the present invention provides a drug-loadedmicelle, as described herein, wherein the drug is 539625.

In certain embodiments, the present invention provides a drug-loadedmicelle, as described herein, wherein the drug is an anthracycline.

Several anthracycline derivates have been produced and have found use inthe clinic for the treatment of leukemias, Hodgkin's lymphoma, as wellas cancers of the bladder, breast, stomach, lung, ovaries, thyroid, andsoft tissue sarcoma. Such anthracycline derivatives include daunorubicin(also known as Daunomycin or daunomycin cerubidine), doxorubicin (alsoknown as DOX, hydroxydaunorubicin, or adriamycin), epirubicin (alsoknown as Ellence or Pharmorubicin), idarubicin (also known as4-demethoxydaunorubicin, Zavedos, or Idamycin), and valrubicin (alsoknown as N-trifluoroacetyladriamycin-14-valerate or Valstar).Anthracyclines are typically prepared as an ammonium salt (e.g.hydrochloride salt) to improve water solubility and allow for ease ofadministration.

In certain embodiments, the present invention provides a drug-loadedmicelle, as described herein, wherein the drug is daunorubicin.

In certain embodiments, the present invention provides a drug-loadedmicelle, as described herein, wherein the drug is doxorubicin.

Aminopterin is well known in the literature and is an analog of folicacid that is an antineoplastic agent. Aminopterin works as an enzymeinhibitor by competing for the folate binding sight of the enzymedihydofolate reductase. The structure of aminopterin is shown below.

In certain embodiments, the present invention provides a drug-loadedmicelle, as described herein, wherein the drug is aminopterin.

Platinum based therapeutics are well known in the literature. Platinumtherapeutics are widely used in oncology and act to crosslink DNA whichresults in cell death (apoptosis). Carboplatin, picoplatin, cisplatin,and oxaliplatin are exemplary platinum therapeutics and the structuresare shown below.

In certain embodiments, the present invention provides a drug-loadedmicelle, as described herein, wherein the drug is picoplatin.

In certain embodiments, the present invention provides a drug-loadedmicelle, as described herein, wherein the drug is a platinumtherapeutic.

Small molecule drugs suitable for loading into micelles of the presentinvention are well known in the art. In certain embodiments, the presentinvention provides a drug-loaded micelle as described herein, whereinthe drug is a hydrophobic drug selected from analgesics,anti-inflammatory agents, HDAC inhibitors, mitotic inhibitors,microtubule stabilizers, DNA intercalators, topoisomerase inhibitors,antihelminthics, anti-arrhythmic agents, anti-bacterial agents,anti-viral agents, anti-coagulants, anti-depressants, anti-diabetics,anti-epileptics, anti-fungal agents, anti-gout agents, anti-hypertensiveagents, anti-malarials, anti-migraine agents, anti-muscarinic agents,anti-neoplastic agents, erectile dysfunction improvement agents,immunosuppressants, anti-protozoal agents, anti-thyroid agents,anxiolytic agents, sedatives, hypnotics, neuroleptics, β-blockers,cardiac inotropic agents, corticosteroids, diuretics, anti-parkinsonianagents, gastro-intestinal agents, histamine receptor antagonists,keratolyptics, lipid regulating agents, anti-anginal agents, Cox-2inhibitors, leukotriene inhibitors, macrolides, muscle relaxants,nutritional agents, opiod analgesics, protease inhibitors, sex hormones,stimulants, muscle relaxants, anti-osteoporosis agents, anti-obesityagents, cognition enhancers, anti-urinary incontinence agents,anti-benign prostate hypertrophy agents, essential fatty acids,non-essential fatty acids, and mixtures thereof.

In other embodiments, the hydrophobic drug is selected from one or moreanalgesics, anti-bacterial agents, anti-viral agents, anti-inflammatoryagents, anti-depressants, anti-diabetics, anti-epileptics,anti-hypertensive agents, anti-migraine agents, immunosuppressants,anxiolytic agents, sedatives, hypnotics, neuroleptics, β-blockers,gastro-intestinal agents, lipid regulating agents, anti-anginal agents,Cox-2 inhibitors, leukotriene inhibitors, macrolides, muscle relaxants,opioid analgesics, protease inhibitors, sex hormones, cognitionenhancers, anti-urinary incontinence agents, and mixtures thereof.

According to one aspect, the present invention provides a micelle, asdescribed herein, loaded with a hydrophobic drug selected from any oneor more of a Exemestance (aromasin), Camptosar (irinotecan), Ellence(epirubicin), Femara (Letrozole), Gleevac (imatinib mesylate), Lentaron(formestane), Cytadren/Orimeten (aminoglutethimide), Temodar, Proscar(finasteride), Viadur (leuprolide), Nexavar (Sorafenib), Kytril(Granisetron), Taxotere (Docetaxel), Taxol (paclitaxel), Kytril(Granisetron), Vesanoid (tretinoin) (retin A), XELODA (Capecitabine),Arimidex (Anastrozole), Casodex/Cosudex (Bicalutamide), Faslodex(Fulvestrant), Iressa (Gefitinib), Nolvadex, Istubal, Valodex (tamoxifencitrate), Tomudex (Raltitrexed), Zoladex (goserelin acetate), Leustatin(Cladribine), Velcade (bortezomib), Mylotarg (gemtuzumab ozogamicin),Alimta (pemetrexed), Gemzar (gemcitabine hydrochloride), Rituxan(rituximab), Revlimid (lenalidomide), Thalomid (thalidomide), Alkeran(melphalan), and derivatives thereof.

E. Crosslinking Chemistries

In certain embodiments, the present invention provides crosslinkedmicelles which effectively encapsulate hydrophobic or ionic therapeuticagents at pH 7.4 (blood) but dissociate and release the drug attargeted, acidic pH values ranging from 5.0 (endosomal pH) to 6.8(extracellular tumor pH). In yet other embodiments, the pH value can beadjusted between 4.0 and 7.4. These pH-targeted nanovectors willdramatically improve the cancer-specific delivery of chemotherapeuticagents and minimize the harmful side effects commonly encountered withpotent chemotherapy drugs. In addition, the utilization of chemistrieswhich can be tailored to dissociate across a range of pH values makethese drug-loaded micelles applicable in treating solid tumors andmalignancies that have become drug resistant.

In certain embodiments, the present invention provides a drug loadedmicelle comprising a triblock copolymer, wherein said micelle has adrug-loaded inner core, a crosslinked outer core, and a hydrophilicshell, wherein the triblock copolymer is of formula VI:

-   -   wherein each of Q, J, T, x, y, n, R^(x), R^(y) and R² is as        defined above and as described in classes and subclasses herein,        both singly and in combination;        -   M is a metal ion;        -   Each R^(T) independently selected from either -J-T or            —Z(CH₂CH₂Y)_(p)(CH₂)_(t)R³, wherein:            -   Z is —O—, —S—, —C≡C—, or —CH₂—;            -   each Y is independently —O— or —S—;            -   p is 0-10;            -   t is 0-10; and        -   R³ is —N₃, —CN, a mono-protected amine, a di-protected            amine, a protected aldehyde, a protected hydroxyl, a            protected carboxylic acid, a protected thiol, a 9-30            membered crown ether, or an optionally substituted group            selected from aliphatic, a 5-8 membered saturated, partially            unsaturated, or aryl ring having 0-4 heteroatoms            independently selected from nitrogen, oxygen, or sulfur, an            8-10 membered saturated, partially unsaturated, or aryl            bicyclic ring having 0-5 heteroatoms independently selected            from nitrogen, oxygen, or sulfur, or a detectable moiety;        -   Q is a valence bond or a bivalent, saturated or unsaturated,            straight or branched C₁₋₁₂ hydrocarbon chain, wherein 0-6            methylene units of Q are independently replaced by -Cy-,            —O—, —NH—, —S—, —OC(O)—, —C(O)O—, —C(O)—, —SO—, —SO₂—,            —NHSO₂—, —SO₂NH—, —NHC(O)—, —C(O)NH—, —OC(O)NH—, or            —NHC(O)O—, wherein:            -   -Cy- is an optionally substituted 5-8 membered bivalent,                saturated, partially unsaturated, or aryl ring having                0-4 heteroatoms independently selected from nitrogen,                oxygen, or sulfur, or an optionally substituted 8-10                membered bivalent saturated, partially unsaturated, or                aryl bicyclic ring having 0-5 heteroatoms independently                selected from nitrogen, oxygen, or sulfur;

In certain embodiments, M is iron. In other embodiments, M is zinc. Inanother embodiment, M is nickel, cobalt, copper, or platinum. In otherembodiments, M is calcium or aluminum. In yet other embodiments, M isstrontium, manganese, platinum, palladium, silver, gold, cadmium,chromium, indium, or lead.

In certain embodiments, the present invention provides a drug loadedmicelle comprising a triblock copolymer, wherein said micelle has adrug-loaded inner core, a crosslinked outer core, and a hydrophilicshell, wherein the triblock copolymer is of formula VII:

-   -   wherein each of Q, J, T, M, m, y, n, R^(y) and R^(T) is as        defined above and as described in classes and subclasses herein,        both singly and in combination;        -   x¹ is 1-20 and;        -   x² is 0-20.

In certain embodiments, the present invention provides a drug loadedmicelle comprising a triblock copolymer, wherein said micelle has adrug-loaded inner core, a crosslinked outer core, and a hydrophilicshell, wherein the triblock copolymer is of formula VIII:

-   -   wherein each of Q, J, T, M, m, y, x¹, x², n, R^(y) and R^(T) is        as defined above and as described in classes and subclasses        herein, both singly and in combination.

In certain embodiments, the present invention provides a drug loadedmicelle comprising a triblock copolymer, wherein said micelle has adrug-loaded inner core, a crosslinked outer core, and a hydrophilicshell, wherein the triblock copolymer is of formula IX:

-   -   wherein each of M, x¹, x², and n is as defined above and as        described in classes and subclasses herein, both singly and in        combination;        -   y¹ is 5-30 and;        -   y² is 10-40.

It will be obvious to one skilled in the art that the drug loaded,crosslinked micelle of the present invention is comprised of tens tohundreds of polymer chains. Despite the fact that only two polymerchains linked by a metal ion is depicted in any of Formula VI, VII,VIII, or IX, it will be understood that the polymer micelle is comprisedof many more polymer chains that are not depicted for ease ofpresentation.

In other embodiments, the present invention provides a system comprisinga triblock copolymer of formula I, a hydrophobic therapeutic agent, anda metal ion. In another embodiment, the present invention provides asystem comprising a triblock copolymer of any of formulae I, II, III,and IV, either singly or in combination, a hydrophobic therapeuticagent, and a metal ion. In yet another embodiment, the present inventionprovides a system comprising a triblock copolymer of formula II, ahydrophobic therapeutic agent, and a metal ion.

In other embodiments, the present invention provides a system comprisinga triblock copolymer of formula VI and a hydrophobic therapeutic agent.In another embodiment, the present invention provides a systemcomprising a triblock copolymer of any of formulae VI, VII, VIII, andIX, either singly or in combination, and a hydrophobic therapeuticagent. In yet another embodiment, the present invention provides asystem comprising a triblock copolymer of formula VII and a hydrophobictherapeutic agent. In some embodiments, the present invention provides asystem comprising a triblock copolymer of formula XI and a hydrophobictherapeutic agent.

The ultimate goal of metal-mediated crosslinking is to ensure micellestability when diluted in the blood (pH 7.4) followed by rapiddissolution and drug release in response to a finite pH change such asthose found in a tumor environment.

In one aspect of the invention, a drug-loaded micelle is crosslinked viaa hydroxamic acid moiety. Hydroxamic acids as described above chelatecertain metals as described in Rosthauser et. al. Macromolecules 1981,14, 538-543 and in Miller Chemical Reviews 1989, 89, 1563-1579(hereinafter “Miller”). This chelation chemistry is shown in Scheme 1.

Accordingly, the addition of a metal ion to a drug loaded micelle of thepresent invention would result in the chelation of the metal ions by thehydroxamic acid, affording a crosslinked, drug loaded micelle. Metalions are selected from, but not limited to: iron, nickel, cobalt, zinc,calcium, copper, strontium, platinum, palladium, vanadium, manganese,and titanium.

One skilled in the art will recognize that the M group of Formula VI,VII, or VIII may be either a divalent or trivalent metal ion. It is alsorecognized that the structures of Formula VI, VII, or VIII, for clarity,are represented using a divalent metal ion. In the case of a trivalentmetal ion as described in Scheme 1, it is understood that there may bethree hydroxamic acid or catechol groups bound to a single metal ion.

In one aspect of the invention, a drug-loaded micelle is crosslinked viaa catechol moiety. Catechols, as described above, complex metal ions asrepresented in Scheme 2. The chelation of catechols with metal ions isalso described in Miller. Accordingly, the addition of a metal ion to adrug loaded micelle of the present invention would result in thechelation of the metal ions by the hydroxamic acid, affording acrosslinked, drug loaded micelle. Metal ions are selected from, but notlimited to: iron, nickel, cobalt, zinc, calcium, copper, strontium,vanadium, manganese, and titanium.

In certain embodiments, the present invention provides a crosslinked,drug-loaded micelle, as described herein, wherein the polymer is

In certain embodiments, the present invention provides a crosslinked,drug-loaded micelle, as described herein, wherein the polymer is

In certain embodiments, the present invention provides a crosslinked,drug-loaded micelle, as described herein, wherein the polymer is

In certain embodiments, the present invention provides a crosslinked,drug-loaded micelle, as described herein, wherein the drug is a taxane.

In certain embodiments, the present invention provides a crosslinked,drug-loaded micelle, as described herein, wherein the drug ispaclitaxel.

In certain embodiments, the present invention provides a crosslinked,drug-loaded micelle, as described herein, wherein the drug is docetaxel.

In certain embodiments, the present invention provides a crosslinked,drug-loaded micelle, as described herein, wherein the drug iscabazitaxel.

In certain embodiments, the present invention provides a crosslinked,drug-loaded micelle, as described herein, wherein the drug is anepothilone.

In certain embodiments, the present invention provides a crosslinked,drug-loaded micelle, as described herein, wherein the drug is EpothiloneB or Epothilone D.

In certain embodiments, the present invention provides a crosslinked,drug-loaded micelle, as described herein, wherein the drug is EpothiloneA or Epothilone C.

In certain embodiments, the present invention provides a crosslinked,drug-loaded micelle, as described herein, wherein the drug is a vincaalkaloid.

In certain embodiments, the present invention provides a crosslinked,drug-loaded micelle, as described herein, wherein the drug isvinorelbine.

In certain embodiments, the present invention provides a crosslinked,drug-loaded micelle, as described herein, wherein the drug is berberine.

In certain embodiments, the present invention provides a crosslinked,drug-loaded micelle, as described herein, wherein the drug isberberrubine.

In certain embodiments, the present invention provides a crosslinked,drug-loaded micelle, as described herein, wherein the drug is acamptothecin.

In certain embodiments, the present invention provides a crosslinked,drug-loaded micelle, as described herein, wherein the drug is SN-38.

In certain embodiments, the present invention provides a crosslinked,drug-loaded micelle, as described herein, wherein the drug is S39625.

In certain embodiments, the present invention provides a crosslinked,drug-loaded micelle, as described herein, wherein the drug is ananthracycline.

In certain embodiments, the present invention provides a crosslinked,drug-loaded micelle, as described herein, wherein the drug isdaunorubicin.

In certain embodiments, the present invention provides a crosslinked,drug-loaded micelle, as described herein, wherein the drug isdoxorubicin.

In certain embodiments, the present invention provides a crosslinked,drug-loaded micelle, as described herein, wherein the drug isaminopterin.

In certain embodiments, the present invention provides a crosslinked,drug-loaded micelle, as described herein, wherein the drug ispicoplatin.

In certain embodiments, the present invention provides a crosslinked,drug-loaded micelle, as described herein, wherein the drug is a platinumtherapeutic.

4. General Methods for Providing Compounds of the Present Invention

Multiblock copolymers of the present invention are prepared by methodsknown to one of ordinary skill in the art. Generally, such multiblockcopolymers are prepared by sequentially polymerizing one or more cyclicamino acid monomers onto a hydrophilic polymer having a terminal aminewherein said polymerization is initiated by said amine. In certainembodiments, said polymerization occurs by ring-opening polymerizationof the cyclic amino acid monomers. In other embodiments, the cyclicamino acid monomer is an amino acid NCA, lactam, or imide.

Scheme 3 above depicts a general method for preparing multiblockpolymers of the present invention. A macroinitiator of formula A istreated with a first amino acid NCA to form a compound of formula Bhaving a first amino acid block. The second amino acid NCA is added tothe living polymer of formula B to give a triblock copolymer of FormulaC having two different amino acid blocks. Each of the R¹, R², n, Q,R^(x), R^(y), x, and y groups depicted in Scheme 3 are as defined anddescribed in classes and subclasses, singly and in combination, herein.

One step in the preparation of a compound of formula I comprisesterminating the living polymer chain-end of the compound of formula Cwith a polymerization terminator to afford a compound of formula I. Oneof ordinary skill in the art would recognize that the polymerizationterminator provides the R² group of formula I. Accordingly, embodimentsdirected to the R² group of formula I as set forth above and herein, arealso directed to the polymerization terminator itself, and similarly,embodiments directed to the polymerization terminator, as set forthabove and herein, are also directed to the R² group of formula I.

As described above, compounds of formula I are prepared from compoundsof formula C by treatment with a terminating agent. One of ordinaryskill in the art would recognize that compounds of formula I are alsoreadily prepared directly from compounds of formula C. One of ordinaryskill in the art would also recognize that the above method forpreparing a compound of formula I may be performed as a “one-pot”synthesis of compounds of formula I that utilizes the living polymerchain-end to incorporate the R² group of formula I. Alternatively,compounds of formula I may also be prepared in a multi-step fashion. Forexample, the living polymer chain-end of a compound of formula C may bequenched to afford an amino group which may then be further derivatized,according to known methods, to afford a compound of formula I.

One of ordinary skill in the art will recognize that a variety ofpolymerization terminating agents are for the present invention. Suchpolymerization terminating agents include any R²-containing groupcapable of reacting with the living polymer chain-end of a compound offormula C, or the free-based amino group of formula C, to afford acompound of formula I. Thus, polymerization terminating agents includeanhydrides, and other acylating agents, and groups that contain aleaving group LG that is subject to nucleophilic displacement.

Alternatively, compounds of formula C may be coupled to carboxylicacid-containing groups to form an amide thereof. Thus, it iscontemplated that the amine group of formula C may be coupled with acarboxylic acid moiety to afford compounds of formula I wherein R² is—NHC(O)R⁴. Such coupling reactions are well known in the art. In certainembodiments, the coupling is achieved with a coupling reagent. Suchreagents are well known in the art and include, for example, DCC andEDC, among others. In other embodiments, the carboxylic acid moiety isactivated for use in the coupling reaction. Such activation includesformation of an acyl halide, use of a Mukaiyama reagent, and the like.These methods, and others, are known to one of ordinary skill in theart, e.g., see, “Advanced Organic Chemistry,” Jerry March, 5^(th) Ed.,pp. 351-357, John Wiley and Sons, N.Y.

A “suitable leaving group that is subject to nucleophilic displacement”is a chemical group that is readily displaced by a desired incomingchemical moiety. Suitable leaving groups are well known in the art,e.g., see, March. Such leaving groups include, but are not limited to,halogen, alkoxy, sulphonyloxy, optionally substituted alkylsulphonyloxy,optionally substituted alkenylsulfonyloxy, optionally substitutedarylsulfonyloxy, and diazonium moieties. Examples of suitable leavinggroups include chloro, iodo, bromo, fluoro, methanesulfonyloxy(mesyloxy), tosyloxy, triflyloxy, nitro-phenyl sulfonyloxy (nosyloxy),and bromo-phenyl sulfonyloxy (brosyloxy).

According to an alternate embodiment, the leaving group may be generatedin situ within the reaction medium. For example, a leaving group may begenerated in situ from a precursor of that compound wherein saidprecursor contains a group readily replaced by said leaving group insitu.

Alternatively, when the R² group of formula I is a mono- or di-protectedamine, the protecting group(s) is removed and that functional group maybe derivatized or protected with a different protecting group. It willbe appreciated that the removal of any protecting group of the R² groupof formula I is performed by methods for that protecting group. Suchmethods are described in detail in Green.

In other embodiments, the R² group of formula I is incorporated byderivatization of the amino group of formula C via anhydride coupling,optionally in the presence of base as appropriate. One of ordinary skillin the art would recognize that anhydride polymerization terminatingagents containing an azide, an aldehyde, a hydroxyl, an alkyne, andother groups, or protected forms thereof, may be used to incorporatesaid azide, said aldehyde, said protected hydroxyl, said alkyne, andother groups into the R² group of compounds of formula I. It will alsobe appreciated that such anhydride polymerization terminating agents arealso suitable for terminating the living polymer chain-end of a compoundof formula C, or freebase thereof. Such anhydride polymerizationterminating agents include, but are not limited to, those set forth inTable 3 below.

TABLE 3 Representative Anhydride Polymerization Terminating Agents

A-1

A-2

A-3

A-4

A-5

A-6

A-7

A-8

A-9

A-10

A-11

A-12

A-13

A-14

A-15

A-16

In certain embodiments, the hydrophilic polymer block is poly(ethyleneglycol) (PEG) having a terminal amine (“PEG macroinitiator”). This PEGmacroinitiator initiates the polymerization of NCAs to provide themultiblock copolymers of the present invention. Such synthetic polymershaving a terminal amine group are known in the art and includePEG-amines. PEG-amines may be obtained by the deprotection of a suitablyprotected PEG-amine. Preparation of such suitably protected PEG-amines,and methods of deprotecting the same, is described in detail in U.S.patent application Ser. No. 11/256,735, filed Oct. 24, 2005 andpublished as US 20060142506 on Jun. 29, 2006, the entirety of which ishereby incorporated herein by reference.

As described in US 20060142506, suitably protected PEG-amines may beformed by terminating the living polymer chain end of a PEG with aterminating agent that contains a suitably protected amine. Accordingly,in other embodiments, the terminating agent has suitably protected aminogroup wherein the protecting group is acid-labile.

Alternatively, synthetic polymers having a terminal amine may beprepared from synthetic polymers that contain terminal functional groupsthat may be converted to amines by known synthetic routes. In certainembodiments, the conversion of the terminal functional groups to theamine is conducted in a single synthetic step. In other embodiments, theconversion of the terminal functional groups to the amine is achieved byway of a multi-step sequence. In yet another embodiment, a protectedamine initiator can be used to polymerize ethylene oxide then terminatedwith an appropriate functional group to form the R¹ group of Formula I.The protected amine initiator can then be deprotected to afford the freeamine for subsequent polymerization. Functional group transformationsthat afford amines or protected amines are well known in the art andinclude those described in Larock, R. C., “Comprehensive OrganicTransformations,” John Wiley & Sons, New York, 1999.

Scheme 4 above shows one exemplary method for preparing the bifunctionalPEGs used to prepare the multiblock copolymers of the present invention.At step (a), the polymerization initiator E is treated with a base toform F. A variety of bases are suitable for the reaction at step (a).Such bases include, but are not limited to, potassium naphthalenide,diphenylmethyl potassium, triphenylmethyl potassium, and potassiumhydride. At step (b), the resulting anion is treated with ethylene oxideto form the polymer G. Polymer G is then quenched with a terminationagent in step (c) to form the R¹ group of polymer H. Exemplarytermination agents for Polymer G can be found in Table 4. Polymer H canbe transformed at step (d) to a compound of formula A by deprotectingthe dibenzyl amine group by hydrogenation.

TABLE 4 Exemplary PEG Termination Agents

D-1

D-2

D-3

D-4

D-5

D-6

D-7

D-8

D-9

D-10

D-11

D-12

According to another embodiment, the present invention provides a methodfor preparing a micelle comprising a multiblock copolymer whichcomprises a polymeric hydrophilic block, optionally a crosslinkable orcrosslinked poly(amino acid block), and a hydrophobic poly(amino acid)block, characterized in that said micelle has an inner core, anoptionally crosslinkable or crosslinked outer core, and a hydrophilicshell, said method comprising the steps of:

-   (a) providing a multiblock copolymer of formula I:

wherein each of the R¹, R², Q, R^(x), R^(y), n, x, and y groups offormula I, are as described in various classes and subclasses, bothsingly and in combination, herein,

-   (b) combining said compound of formula I with a therapeutic agent;    and-   (c) treating the resulting micelle with a crosslinking reagent to    crosslink R^(x).

In one embodiment, drugs are loaded into the micelle inner core byadding an aliquot of a copolymer solution in water to the drug to beincorporated. For example, a stock solution of the drug in a polarorganic solvent is made and allowed to evaporate, and then thecopolymer/water solution is added. In another embodiment, the drug isincorporated using an oil in water emulsion technique. In this case, thedrug is dissolved in an organic solvent and added dropwise to themicelle solution in water, and the drug is incorporated into the micelleduring solvent evaporation. In another embodiment, the drug is dissolvedwith the copolymer in a common polar organic solvent and dialyzedagainst water or another aqueous medium. See Allen, C.; Maysinger, D.;Eisenberg A. Colloid Surface B 1999, 16, 3-27.

5. Uses, Methods, and Compositions

As described herein, micelles of the present invention can encapsulate awide variety of therapeutic agents useful for treating a wide variety ofdiseases. In certain embodiments, the present invention provides adrug-loaded micelle, as described herein, wherein said micelle is usefulfor treating the disorder for which the drug is known to treat.According to one embodiment, the present invention provides a method fortreating one or more disorders selected from pain, inflammation,arrhythmia, arthritis (rheumatoid or osteoarthritis), atherosclerosis,restenosis, bacterial infection, viral infection, depression, diabetes,epilepsy, fungal infection, gout, hypertension, malaria, migraine,cancer or other proliferative disorder, erectile dysfunction, a thyroiddisorder, neurological disorders and hormone-related diseases,Parkinson's disease, Huntington's disease, Alzheimer's disease, agastro-intestinal disorder, allergy, an autoimmune disorder, such asasthma or psoriasis, osteoporosis, obesity and comorbidities, acognitive disorder, stroke, AIDS-associated dementia, amyotrophiclateral sclerosis (ALS, Lou Gehrig's disease), multiple sclerosis (MS),schizophrenia, anxiety, bipolar disorder, tauopothy, a spinal cord orperipheral nerve injury, myocardial infarction, cardiomyocytehypertrophy, glaucoma, an attention deficit disorder (ADD or ADHD), asleep disorder, reperfusion/ischemia, an angiogenic disorder, or urinaryincontinence, comprising administering to a patient a micelle comprisinga multiblock copolymer which comprises a polymeric hydrophilic block,optionally a crosslinkable or crosslinked poly(amino acid block), and ahydrophobic D,L-mixed poly(amino acid block), characterized in that saidmicelle has a drug-loaded inner core, optionally a crosslinkable orcrosslinked outer core, and a hydrophilic shell, wherein said micelleencapsulates a therapeutic agent suitable for treating said disorder.

In other embodiments, the present invention provides a method fortreating one or more disorders selected from autoimmune disease, aninflammatory disease, a metabolic disorder, a psychiatric disorder,diabetes, an angiogenic disorder, tauopothy, a neurological orneurodegenerative disorder, a spinal cord injury, glaucoma, baldness, ora cardiovascular disease, comprising administering to a patient amultiblock copolymer which comprises a polymeric hydrophilic block,optionally a crosslinkable or crosslinked poly(amino acid block), and ahydrophobic D,L-mixed poly(amino acid block), characterized in that saidmicelle has a drug-loaded inner core, optionally a crosslinkable orcrosslinked outer core, and a hydrophilic shell, wherein said micelleencapsulates a therapeutic agent suitable for treating said disorder.

In certain embodiments, drug-loaded micelles of the present inventionare useful for treating cancer. Accordingly, another aspect of thepresent invention provides a method for treating cancer in a patientcomprising administering to a patient a multiblock copolymer whichcomprises a polymeric hydrophilic block, optionally a crosslinkable orcrosslinked poly(amino acid block), and a hydrophobic D,L-mixedpoly(amino acid block), characterized in that said micelle has adrug-loaded inner core, optionally a crosslinkable or crosslinked outercore, and a hydrophilic shell, wherein said micelle encapsulates achemotherapeutic agent. According to another embodiment, the presentinvention relates to a method of treating a cancer selected from breast,ovary, cervix, prostate, testis, genitourinary tract, esophagus, larynx,glioblastoma, neuroblastoma, stomach, skin, keratoacanthoma, lung,epidermoid carcinoma, large cell carcinoma, small cell carcinoma, lungadenocarcinoma, bone, colon, adenoma, pancreas, adenocarcinoma, thyroid,follicular carcinoma, undifferentiated carcinoma, papillary carcinoma,seminoma, melanoma, sarcoma, bladder carcinoma, liver carcinoma andbiliary passages, kidney carcinoma, myeloid disorders, lymphoiddisorders, Hodgkin's, hairy cells, buccal cavity and pharynx (oral),lip, tongue, mouth, pharynx, small intestine, colon-rectum, largeintestine, rectum, brain and central nervous system, and leukemia,comprising administering a micelle in accordance with the presentinvention wherein said micelle encapsulates a chemotherapeutic agentsuitable for treating said cancer.

P-glycoprotein (Pgp, also called multidrug resistance protein) is foundin the plasma membrane of higher eukaryotes where it is responsible forATP hydrolysis-driven export of hydrophobic molecules. In animals, Pgpplays an important role in excretion of and protection fromenvironmental toxins, when expressed in the plasma membrane of cancercells, it can lead to failure of chemotherapy by preventing thehydrophobic chemotherapeutic drugs from reaching their targets insidecells. Indeed, Pgp is known to transport hydrophobic chemotherapeuticdrugs out of tumor cells. According to one aspect, the present inventionprovides a method for delivering a hydrophobic chemotherapeutic drug toa cancer cell while preventing, or lessening, Pgp excretion of thatchemotherapeutic drug, comprising administering a drug-loaded micellecomprising a multiblock polymer of the present invention loaded with ahydrophobic chemotherapeutic drug. Such hydrophobic chemotherapeuticdrugs are well known in the art and include those described herein.

Compositions

According to another embodiment, the invention provides a compositioncomprising a micelle of this invention or a pharmaceutically acceptablederivative thereof and a pharmaceutically acceptable carrier, adjuvant,or vehicle. In certain embodiments, the composition of this invention isformulated for administration to a patient in need of such composition.In other embodiments, the composition of this invention is formulatedfor oral administration to a patient.

The term “patient”, as used herein, means an animal, preferably amammal, and most preferably a human.

The term “pharmaceutically acceptable carrier, adjuvant, or vehicle”refers to a non-toxic carrier, adjuvant, or vehicle that does notdestroy the pharmacological activity of the compound with which it isformulated. Pharmaceutically acceptable carriers, adjuvants or vehiclesthat may be used in the compositions of this invention include, but arenot limited to, ion exchangers, alumina, aluminum stearate, lecithin,serum proteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of acid salts include acetate,adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate,butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate,glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate,hexanoate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oxalate, palmoate,pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate,propionate, salicylate, succinate, sulfate, tartrate, thiocyanate,tosylate and undecanoate. Other acids, such as oxalic, while not inthemselves pharmaceutically acceptable, may be employed in thepreparation of salts useful as intermediates in obtaining the compoundsof the invention and their pharmaceutically acceptable acid additionsalts.

Salts derived from appropriate bases include alkali metal (e.g., sodiumand potassium), alkaline earth metal (e.g., magnesium), ammonium andN+(C1-4 alkyl)4 salts. This invention also envisions the quaternizationof any basic nitrogen-containing groups of the compounds disclosedherein. Water or oil-soluble or dispersible products may be obtained bysuch quaternization.

The compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,intraperitoneally or intravenously. Sterile injectable forms of thecompositions of this invention may be aqueous or oleaginous suspension.These suspensions may be formulated according to techniques known in theart using dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a non-toxic parenterally acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium.

For this purpose, any bland fixed oil may be employed includingsynthetic mono- or di-glycerides. Fatty acids, such as oleic acid andits glyceride derivatives are useful in the preparation of injectables,as are natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as carboxymethyl cellulose or similar dispersingagents that are commonly used in the formulation of pharmaceuticallyacceptable dosage forms including emulsions and suspensions. Othercommonly used surfactants, such as Tweens, Spans and other emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

The pharmaceutically acceptable compositions of this invention may beorally administered in any orally acceptable dosage form including, butnot limited to, capsules, tablets, aqueous suspensions or solutions. Inthe case of tablets for oral use, carriers commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added. In certain embodiments,pharmaceutically acceptable compositions of the present invention areenterically coated.

Alternatively, the pharmaceutically acceptable compositions of thisinvention may be administered in the form of suppositories for rectaladministration. These can be prepared by mixing the agent with asuitable non-irritating excipient that is solid at room temperature butliquid at rectal temperature and therefore will melt in the rectum torelease the drug. Such materials include cocoa butter, beeswax andpolyethylene glycols.

The pharmaceutically acceptable compositions of this invention may alsobe administered topically, especially when the target of treatmentincludes areas or organs readily accessible by topical application,including diseases of the eye, the skin, or the lower intestinal tract.Suitable topical formulations are readily prepared for each of theseareas or organs.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a enema formulation.Topically-transdermal patches may also be used.

For topical applications, the pharmaceutically acceptable compositionsmay be formulated in an ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutically acceptable compositions canbe formulated in a lotion or cream containing the active componentssuspended or dissolved in one or more pharmaceutically acceptablecarriers. Suitable carriers include, but are not limited to, mineraloil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearylalcohol, 2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutically acceptable compositions may beformulated as micronized suspensions in isotonic, pH adjusted sterilesaline, or, preferably, as solutions in isotonic, pH adjusted sterilesaline, either with or without a preservative such as benzylalkoniumchloride. Alternatively, for ophthalmic uses, the pharmaceuticallyacceptable compositions may be formulated in an ointment such aspetrolatum.

The pharmaceutically acceptable compositions of this invention may alsobe administered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

In certain embodiments, the pharmaceutically acceptable compositions ofthis invention are formulated for oral administration.

The amount of the compounds of the present invention that may becombined with the carrier materials to produce a composition in a singledosage form will vary depending upon the host treated, the particularmode of administration. Preferably, the compositions should beformulated so that a dosage of between 0.01-100 mg/kg body weight/day ofthe drug can be administered to a patient receiving these compositions.

It will be appreciated that dosages typically employed for theencapsulated drug are contemplated by the present invention. In certainembodiments, a patient is administered a drug-loaded micelle of thepresent invention wherein the dosage of the drug is equivalent to whatis typically administered for that drug. In other embodiments, a patientis administered a drug-loaded micelle of the present invention whereinthe dosage of the drug is lower than is typically administered for thatdrug.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a compound of the present invention in the composition willalso depend upon the particular compound in the composition.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It will be understoodthat these examples are for illustrative purposes only and are not to beconstrued as limiting this invention in any manner.

EXEMPLIFICATION

As described generally above, multiblock copolymers of the presentinvention are prepared using the heterobifunctional PEGs describedherein and in U.S. patent application Ser. No. 11/256,735, filed Oct.24, 2005, published as WO2006/047419 on May 4, 2006 and published as US20060142506 on Jun. 29, 2006, the entirety of which is herebyincorporated herein by reference. The preparation of multiblock polymersin accordance with the present invention is accomplished by methodsknown in the art, including those described in detail in U.S. patentapplication Ser. No. 11/325,020, filed Jan. 4, 2006, published asWO2006/74202 on Jul. 13, 2006 and published as US 20060172914 on Aug. 3,2006, the entirety of which is hereby incorporated herein by reference.

In each of the Examples below, where an amino acid, or correspondingNCA, is designated “D”, then that amino acid, or corresponding NCA, isof the D-configuration. Where no such designation is recited, then thatamino acid, or corresponding NCA, is of the L-configuration.

General Methods:

Particle Size Analysis

Dynamic light scattering with a Wyatt Dynapro plate reader was used todetermine the particle sizes of the uncrosslinked and crosslinkedformulations. Solutions of the formulations were made at 1 mg/mL in 150mM NaCl. The samples were centrifuged at 2000 RPM for 5 minutes, andthen 300 μL each was added to a well of a 96-well plate in triplicatefor analysis. 10 acquisitions per well with 30-second acquisition timesand laser auto-attenuation were used to collect the data.

Encapsulation Verification Dialysis

The uncrosslinked formulation was dissolved in 3.5 mL of 10 mM phosphatebuffer pH 8 at 20 mg/mL, and at 0.2 mg/mL. 3 mL of the samples was addedto 3500 molecular weight-cutoff dialysis bags, and the remaining 0.5 mLwas added to HPLC vials for the pre dialysis samples. The dialysis bagswere placed in 300 mL of 10 mM PB pH 8 and stirred for 6 hours. Aliquotswere then taken from inside the dialysis bags and HPLC analysis was usedto determine the peak areas of drug from the pre dialysis and postdialysis samples. The areas were then used to calculate the % drugremaining post dialysis.

Iron-Dependent Crosslinking and Analysis

The uncrosslinked formulation was reconstituted in water at 20 mg/mLwith either 0.1, 0.25, 0.5, 0.75, 1, 2.5, 5, 7.5 or 10 mM iron (III)chloride and allowed to stir over night at room temperature. The sampleswere then diluted to 0.2 mg mL in 10 mM phosphate buffer pH 8, with afinal volume of 5 mL. Aliquots of 1.5 mL were taken as pre-dialysissamples for HPLC analysis, then 3 mL of each sample was added to a 3500MWC cut-off dialysis bag and dialyzed against 10 mM phosphate buffer pH8 for 6 hours. After 6 hours the samples were removed from inside thedialysis bags and analyzed by HPLC. The post-dialysis peak area for eachsample was divided by the pre-dialysis peak areas and multiplied by 100to converted to percent remaining.

Time-Dependent Crosslinking and Analysis

The uncrosslinked formulation was reconstituted in water at 20 mg/mL,and 50 μL was diluted into 4.95 mL for the uncrosslinked sample. A 500mM stock solution of iron (III) chloride was then added to theuncrosslinked solution for a final concentration of 10 mM iron (III)chloride. This was used as the stock crosslinked solution, where 50 μLaliquots were taken at 5 minutes, 30 minutes, 1 hour, 2 hours, 4 hoursand 16 hours and diluted to 0.2 mg mL in 10 mM phosphate buffer pH 8,with a final volume of 5 mL. Aliquots of 1.5 mL were taken aspre-dialysis samples for HPLC analysis, then 3 mL of each sample wasadded to a 3500 MWC cut-off dialysis bag and dialyzed against 10 mMphosphate buffer pH 8 for 6 hours. After 6 hours the samples wereremoved from inside the dialysis bags and analyzed by HPLC. Thepost-dialysis peak area for each sample was divided by the pre-dialysispeak areas and multiplied by 100 to converted to percent remaining.

pH-Dependent Crosslinking and Analysis

The uncrosslinked formulation was reconstituted in water at 20 mg/mLwith 10 mM iron (III) chloride at pH 3, 4, 5, 6, 7, 7.4 and 8. Thesamples were allowed to incubate for 10 minutes following reconstitutionand pH adjustment, and then diluted to 0.2 mg mL in 10 mM phosphatebuffer pH 8, with a final volume of 5 mL. Aliquots of 1.5 mL were takenas pre-dialysis samples for HPLC analysis, then 3 mL of each sample wasadded to a 3500 MWC cut-off dialysis bag and dialyzed against 10 mMphosphate buffer pH 8 for 6 hours. After 6 hours the samples wereremoved from inside the dialysis bags and analyzed by HPLC. Thepost-dialysis peak area for each sample was divided by the pre-dialysispeak areas and multiplied by 100 to converted to percent remaining.

pH-Dependent Release of Crosslinked Formulations

The uncrosslinked formulation was reconstituted in water at 20 mg/mLwith 10 mM iron (III) chloride, pH adjusted to 8.0 with NaOH and allowedto stir over night at room temperature. The next day the sample wasdiluted to 0.2 mg/mL in 10 mM phosphate buffer at pH 3, 4, 5, 6, 7, 7.4and 8, with a final volume of 5 mL per sample. Aliquots of 1.5 mL weretaken as pre-dialysis samples for HPLC analysis, then 3 mL of eachsample was added to a 3500 MWC cut-off dialysis bag and dialyzed against10 mM phosphate buffer pH 8 for 6 hours. After 6 hours the samples wereremoved from inside the dialysis bags and analyzed by HPLC. Thepost-dialysis peak area for each sample was divided by the pre-dialysispeak areas and multiplied by 100 to converted to percent remaining.

pH-Dependent Release of Uncrosslinked Formulations

The uncrosslinked formulation was reconstituted in water at 20 mg/mL, pHadjusted to 8.0 with NaOH and allowed to stir over night at roomtemperature. The next day the sample was diluted to 0.2 mg/mL in 10 mMphosphate buffer at pH 3, 4, 5, 6, 7, 7.4 and 8, with a final volume of5 mL per sample. Aliquots of 1.5 mL were taken as pre-dialysis samplesfor HPLC analysis, then 3 mL of each sample was added to a 3500 MWCcut-off dialysis bag and dialyzed against 10 mM phosphate buffer pH 8for 6 hours. After 6 hours the samples were removed from inside thedialysis bags and analyzed by HPLC. The post-dialysis peak area for eachsample was divided by the pre-dialysis peak areas and multiplied by 100to converted to percent remaining.

Salt-Dependent Release of Crosslinked Formulations

The uncrosslinked formulation was reconstituted in water at 20 mg/mLwith 10 mM iron (III) chloride, pH adjusted to 8.0 with NaOH and allowedto stir for 10 minutes. The sample was then diluted to 0.2 mg/mL in 10mM phosphate buffer pH 8 with increasing NaCl concentration from 0 to10, 50, 100, 200, 300, 400 and 500 mM with a final volume of 5 mL persample. Aliquots of 1.5 mL were taken as pre-dialysis samples for HPLCanalysis, then 3 mL of each sample was added to a 3500 MWC cut-offdialysis bag and dialyzed against 10 mM phosphate buffer pH 8 with thecorresponding salt concentration for 6 hours. After 6 hours the sampleswere removed from inside the dialysis bags and analyzed by HPLC. Thepost-dialysis peak area for each sample was divided by the pre-dialysispeak areas and multiplied by 100 to converted to percent remaining.

In-Vitro Cytotoxicity of Aminopterin Formulations

Cells originally purchased from ATCC (A549, Panc-1, OVCAR3, and BXPC-3)were seeded in 96 well tissue culture plates to be 50% confluent by 24hours. Cells were incubated at 37° C. with 5.0% CO₂. Cells were treatedwith escalading doses of free aminopterin, crosslinked aminopterinformulation, uncrosslinked aminopterin formulation, and non drug-loadedmicelle formulations 24 hours after plate seeding. Free aminopterin wasdissolved in DMSO and administered to cells with a total volume of DMSOequal to or less than 0.0025%. Micelle formulations were re-suspended inbiology grade water. Dilutions were done in deep well plates with cellmedia and water or DMSO (for free aminopterin only) equalizing displacedvolume. Incubation media was aspirated from the 96 well plates and 100μl of each dilution was added to the wells in triplicate and incubatedover 72 hours at 37° C. with 5.0% CO₂. Crosslinked and uncrosslinked nondrug-loaded micelle formulations were administered at the four highestdoses and were calculated at comparative mg/ml concentrations todrug-loaded micelle concentrations of delivery vehicle. After 72-hourincubation, plates were allowed to cool to room temperature and 25 μl ofcell titer-glo was added to each well. Plates were briefly shaken to mixand luminescence readings were read on a plate reader. Luminescencereading for triplicate doses are averaged and divided by averageluminescence readings from untreated cells on the same plate tocalculate the % of viable cells per dose.

Formulation Method

A Polymer was dissolved in water at a concentration of 5 mg/mL bystirring and heating to 40 degrees Celsius for approximately 30 minutes.Sucrose was then added to the polymer solution at 5 mg/mL and stirred atroom temperature until homogenous. The solution was then allowed to coolto room temperature while stirring. The active pharmaceutical ingredient(API) was dissolved in organic solvent just below the limit ofsolubility. The API/organic solution was then added to thepolymer/sucrose solution while shear mixing at 10,000 RPM forapproximately 30 seconds, or until a homogenous emulsion resulted. Thesolution was then processed with a single pass through a microfluidizerwith an operating pressure of approximately 23,000 PSI with the outletstream cooled by an ice water bath. The solution was then passed througha 0.22 micron dead-end filter, and then processed by ultrafiltrationusing tangential flow filtration until a total of 4-times the originalvolume of sucrose buffer was exchanged and the final concentration ofpolymer in solution was approximately 20 mg/mL. Iron (III) Chloride wasthen added to the formulation for a final concentration of 10 mM. The pHof the solution was then adjusted to 6.0 with NaOH and stirred at roomtemperature for 4 hours. One volume of buffer containing acryopreservative agent at 20 mg/mL was then added to the solution, andthen concentrated back down to approximately 20 mg/mL polymerconcentration. The solution was then frozen at −40 degrees Celsius andlyophilized.

Formulation Method B

Polymer was dissolved in water at a concentration of 2 mg/mL by stirringand heating to 40 degrees Celsius for approximately 30 minutes. Thesolution was then allowed to cool to room temperature while stirring.The active pharmaceutical ingredient (API) was dissolved in organicsolvent just below the limit of solubility. The API/organic solution wasthen added to the polymer/sucrose solution while shear mixing at 10,000RPM for approximately 30 seconds, or until a homogenous emulsionresulted. The solution was then stirred over night in a fume hood toallow the organic solution to evaporate. The next day the solution waspassed through a 0.22 micron dead-end filter, and then processed byultrafiltration using tangential flow filtration to concentrate thesample from 2 mg/mL to approximately 20 mg/mL. Iron (III) Chloride wasthen added to the formulation for a final concentration of 10 mM. The pHof the solution was then adjusted to 6.0 with NaOH and stirred at roomtemperature for 4 hours. The solution was then frozen at −40 degreesCelsius and lyophilized.

SN-38 Formulation Weight Loading Analysis

Weight loading was determined by comparing a standard curve of SN38 to aknown concentration of formulation by HPLC analysis. SN38 was dissolvedin methanol in a range from 30 μg/mL to 150 μg/mL, and the formulationwas dissolved at 5 mg/mL in methanol. The amount of SN-38 in theformulation is then converted to % based on the known quantity offormulation used (i.e. 5 mg/mL).

Daunorubicin Formulation Weight Loading Analysis

Weight loading was determined by comparing a standard curve ofdaunorubicin to a known concentration of formulation by HPLC analysis.Daunorubicin was dissolved in methanol in a range from 40 μg/mL to 200μg/mL, and the formulation was dissolved at 2 mg/mL in methanol. Theamount of daunorubicin in the formulation is then converted to % basedon the known quantity of formulation used (i.e. 2 mg/mL).

Aminopterin Formulation Weight Loading Analysis

Weight loading was determined by comparing a standard curve ofaminopterin to a known concentration of formulation by HPLC analysis.Aminopterin was dissolved in HPLC mobile phase (60% acetonitrile, 40% 10mM phosphate buffer pH 8) in a range from 40

g/mL to 200

g/mL, and the formulation was dissolved at 5 mg/mL in HPLC mobile phase.The amount of aminopterin in the formulation is then converted to %based on the known quantity of formulation used (i.e. 5 mg/mL).

Berberine Formulation Weight Loading Analysis

Weight loading was determined by comparing a standard curve of berberineto a known concentration of formulation by HPLC analysis. Berberine wasdissolved in methanol in a range from 40 μg/mL to 200 μg/mL, and theformulation was dissolved at 5 mg/mL in methanol. The amount ofberberine in the formulation was then converted to % based on the knownquantity of formulation used (i.e. 5 mg/mL).

Cabazitaxel Formulation Weight Loading Analysis

Weight loading was determined by comparing a standard curve ofcabazitaxel to a known concentration of formulation by HPLC analysis.Cabazitaxel was dissolved in methanol in a range from 40 μg/mL to 200μg/mL, and the formulation was dissolved at 10 mg/mL in methanol. Theamount of cabazitaxel in the formulation was then converted to % basedon the known quantity of formulation used (i.e. 10 mg/mL).

Epothilone D Formulation Weight Loading Analysis

Weight loading was determined by comparing a standard curve ofepothilone D to a known concentration of formulation by HPLC analysis.Epothilone D was dissolved in methanol in a range from 40 μg/mL to 200μg/mL, and the formulation was dissolved at 10 mg/mL in methanol. Theamount of epothilone D in the formulation was then converted to % basedon the known quantity of formulation used (i.e. 10 mg/mL).

General Rat Pharmacokinetic Experiments

Sprague-Dawly rats surgically modified with jugular vein catheters werepurchased from Harlan Laboratories, Dublin, Va. Formulations weredissolved in water with 150 mM NaCl for a final concentration oftypically 10 mg API per kg animal body weight for 1 mL bolus injectionvia JVC over approximately 1 minute, followed by a flush ofapproximately 250

L heparinized saline. Time points for blood collection following testarticle administration were as followed: 1, 5, 15 minutes, 1, 4, 8 and24 hours. Approximately 250 μL of blood per time point was collected byJVC into K3-EDTA blood collection tubes followed by a flush ofapproximately 250 μL heparinized saline. Blood was then centrifuged at2000 RPM for 5 minutes to isolate plasma. Plasma was then collected andsnap frozen until processed for HPLC analysis. Samples were prepared foranalysis by first thawing the plasma samples at room temperature. 50 μLplasma was added to a 2 mL eppendorf tube 150 μL of extraction solution(0.1% phosphoric acid in methanol, 5 μg/mL internal standard). Sampleswere then vortexed for 10 minutes and centrifuged for 10 minutes at13,000 RPM. Supernatant was then transferred into HPLC vials thenanalyzed by HPLC. Quantitation of API was determined using a standardcurve of API formulation in rat plasma compared to samples collectedfrom rats at each time point.

Example 1

Dibenzylamino Ethanol

Benzyl chloride (278.5 g, 2.2 mol), ethanol amine (60 mL, 1 mol),potassium carbonate (283.1 g, 2.05 mol) and ethanol (2 L) were mixedtogether in a 3 L 3-neck flask, fitted with an overhead stirrer, acondenser and a glass plug. The apparatus was heated up to reflux for 36hr, after which the insoluble solid was filtered through a medium frit.The filtrate was recovered and ethanol was removed by rotaryevaporation. The viscous liquid was redissolved in ether, the solidsuspension removed by filtration and extracted twice against water. Theether solution was kept and the aqueous layer was extracted twice withdichloromethane (2×400 mL). The fraction were recombined, dried overMgSO₄, stirred over carbon black for 15 min and filtered through acelite pad. Dichloromethane was removed and the solid was redissolvedinto a minimal amount of ether (combined volume of 300 mL with the firstether fraction, 300 mL). Hexanes (1700 mL) was added and the solutionwas heated up gently till complete dissolution of the product. Thesolution was then cooled down gently, placed in the fridge (+4° C.)overnight and white crystals were obtained. The recrystallization wasdone a second time. 166.63 g, 69% yield. ¹H NMR (d₆-DMSO) δ 7.39-7.24(10H), 4.42 (1H), 3.60 (4H), 3.52 (2H), 2.52 (2H).

Example 2

Dibenzylamino-PEG-methoxy

An apparatus consisting of a 4 L jacketed 3-necked polymerization flaskequipped with a glass magnetic stirring bar and thermally-insulatedjacketed addition funnel was evacuated down to 10 mTorr then backfilledwith argon. The reaction flask was loaded with N,N-dibenzylaminoethanol(4.28 g, 17.7 mmol) and 50% KH solid in paraffin wax (1.70 g, 21.2 mmol)under a gentle stream of argon gas. Anhydrous THF, approximately 2 L,was introduced into the reaction flask and the mixture was stirred underArgon at ambient temperature for 16 h. The resulting slurry was cooledto 10° C., and the addition funnel under vacuum was chilled to −30° C.Ethylene oxide gas was condensed into the chilled evacuated funnel until225 mL (4.8 mol) of liquid EO was collected. The liquid ethylene oxidein the condensation funnel was added in one portion into the reactionmixture. The reaction mixture was stirred in a closed flask at 10° C.for 6 hours, then 20° C. for 16 hours. The polymerization was completedby raising the temperature to 30° C. for 16 hours, then to 40° C. for 2days. The reaction mixture was cooled to 25° C., then methyl iodide (1.6mL) was added at once and the mixture was stirred at 25° C. for 10hours. The excess of unreacted potassium hydride was then destroyed byaddition of ethanol (99%, 100 mL). After 30 min, the quenched reactionmixture was transferred into a large beaker and the polymer product wasprecipitated by addition of ethyl ether (8 L). The precipitated productwas collected by filtration on a large Buchner funnel and then dried invacuo. The yield was 215.1 g of a white solid. Aqueous GPC showed M_(n)of 12.0 kDa and a PDI of 1.01. ¹H-NMR (d₆-DMSO, 400 MHz): 7.344 (m, 8H),7.225 (m, 4H), 3.681 (m, 8H), 3.507 (m, approx. 1000H), 3.320 (m,6H+water signal), 3.237 (s, 3H), 2.551 (t, 6.0 Hz, 2H).

Example 3

mPEG-amine

The mPEG-dibenzylamine product Example 3 (214.0 g) was dissolved indeionized water (1 L). Pearlman's catalyst 13.2 g (20% Pd hydroxide oncarbon, Aldrich) slurry in deionized water (150 mL) was activated bystirring under hydrogen balloon at ambient temperature. The hydrogen inthe flask was replaced with nitrogen, the solution of dibenzylamino mPEGstarting material was added to the catalyst slurry and the flask wasevacuated, then back-filled with hydrogen (repeated 3 times). Thehydrogenation was then continued at ambient temperature under hydrogenballoon for 2½ days at which point ¹H-NMR indicated a completedisappearance of benzyl signals. Sodium chloride (350 g) solid was addedto the reaction mixture and the mixture was stirred for half a day undernitrogen, the spent catalyst was removed by filtration and rinsedthoroughly with brine. The combined filtrates were made alkaline (toapprox pH 11) by addition of a small volume of 1 M NaOH and extractedwith dichloromethane (4×0.7 L). The combined extracts were dried withanhydrous sodium carbonate, filtered and concentrated on rotovap down toabout 0.75 L total volume, then precipitated without a delay by addingexcess of ether (8 L). The precipitated product was collected byfiltration and dried in vacuo to provide 202.5 g of a voluminoussnow-white solid. ¹H-NMR (d₆-DMSO, 400 MHz): 3.681 (m, 8H), 3.507 (m,approx. 1000H), 3.341 (m, 4H+water signal), 3.238 (s, 3H), 2.634 (t, 5.7Hz, 2H).

Example 4

D-Leucine NCA

H-D-Leu-OH (100 g, 0.76 mol) was suspended in 1 L of anhydrous THF andheated to 50° C. while stirring heavily. Phosgene (20% in toluene) (500mL, 1 mol) was added to the amino acid suspension. After 1 h 20 min, theamino acid dissolved, forming a clear solution. The solution wasconcentrated on the rotovap, transferred to a beaker, and hexane wasadded to precipitate the product. The white solid was isolated byfiltration and dissolved in toluene (˜700 mL) with a small amount of THF(˜60 mL). The solution was filtered over a bed of Celite to remove anyinsoluble material. An excess of hexane (˜4 L) was added to the filtrateto precipitate the product. The NCA was isolated by filtration and driedin vacuo. (91 g, 79% yield) D-Leu NCA was isolated as a white,crystalline solid. ¹H NMR (d₆-DMSO) δ 9.13 (1H), 4.44 (1H), 1.74 (1H),1.55 (2H), 0.90 (6H) ppm.

Example 5

tert-Butyl Aspartate NCA

H-Asp(OBu)-OH (120 g, 0.63 mol) was suspended in 1.2 L of anhydrous THFand heated to 50° C. while stirring heavily. Phosgene (20% in toluene)(500 mL, 1 mol) was added to the amino acid suspension. After 1 h 30min, the amino acid dissolved, forming a clear solution. The solutionwas concentrated on the rotovap, transferred to a beaker, and hexane wasadded to precipitate the product. The white solid was isolated byfiltration and dissolved in anhydrous THF. The solution was filteredover a bed of Celite to remove any insoluble material. An excess ofhexane was added to precipitate the product. The NCA was isolated byfiltration and dried in vacuo. 93 g (68%) of Asp(OBu) NCA was isolatedas a white, crystalline solid. ¹H NMR (d₆-DMSO) δ 8.99 (1H), 4.61 (1H),2.93 (1H), 2.69 (1H), 1.38 (9H) ppm.

Example 6

Benzyl Tyrosine NCA

H-Tyr(OBzl)-OH (140 g, 0.52 mol) was suspended in 1.5 L of anhydrous THFand heated to 50° C. while stirring heavily. Phosgene (20% in toluene)(500 mL, 1 mol) was added to the amino acid suspension via cannulation.The amino acid dissolved over the course of approx. 1 h 30, forming apale yellow solution. The solution was first filtered through a Buchnerfitted with a Whatman paper #1 to remove any particles still insuspension. Then, the solution was concentrated by rotary evaporation,transferred to a beaker, and hexane was added to precipitate theproduct. The off-white solid was isolated by filtration and dissolved inanhydrous THF (˜600 mL). The solution was filtered over a bed of Celiteto remove any insoluble material. An excess of hexane (˜6 L) was addedto the filtrate to precipitate the product. The NCA was isolated byfiltration and dried in vacuo. 114.05 g, 74.3% of Tyr(OBzl) NCA wasisolated as an off-white powder. ¹H NMR (d₆-DMSO) δ 9.07 (1H), 7.49-7.29(5H), 7.12-7.07 (2H), 6.98-6.94 (2H), 5.06 (2H), 4.74 (1H), 3.05-2.88(2H) ppm.

Example 7

Phenylalanine NCA

H-L-Phe-OH (20.0 g, 132 mmol) was suspended in 300 mL of anhydrous THFand heated to 50° C. Phosgene (20% in toluene) (90 mL, 182 mmol) wasadded to the amino acid suspension, and the amino acid dissolved overthe course of approx. 1 hr, forming a cloudy solution. The solution wasfiltered through a paper filter (Whatman #1), concentrated on by rotaryevaporation, transferred to a beaker, and hexane was added toprecipitate the product. The white solid was isolated by filtration anddissolved in anhydrous THF. The solution was filtered over a bed ofCelite to remove any insoluble material. An excess of hexanes were addedon the filtrate while stirring with a spatula. The NCA was isolated byfiltration and dried in vacuo. 20.0 g (86% yield) of D-PheNCA wasisolated as a white, crystalline solid. ¹H NMR (d₆-DMSO) δ 9.09 (1H),7.40-7.08 (5H), 4.788 (1H), 3.036 (2H) ppm.

Example 8

L-benzylglutamate NCA

Vacuum-dried H-Glu(OBn)-OH (71.2 g, 300.0 mmol) was suspended in 900 mLof anhydrous THF. Phosgene (20% in toluene) (210 mL, 420 mmol) was addedto the amino acid suspension at room temperature and after ten minutes,the mixture was heated to 50° C. The amino acid dissolved over thecourse of approx. 1 hr, forming a clear solution. The solution wasslightly cooled and concentrated on the rotovap. Fresh anhydrous THF(400 mL) was added to the residue and the solution was re-evaporated onthe rotovap to give a colorless solid, which was dissolved in 300 mLanhydrous THF, transferred to a 4 L beaker and precipitated by the slowaddition of 1.5 L of anhydrous heptane. The pure NCA was isolated bysuction filtration and dried in vacuo. 75.31 g (95.4% yield) of Glu(OBn)NCA was isolated as a colorless, crystalline solid. ¹H NMR (CDCl₃) δ7.36 (5H), 6.40 (1H), 5.14 (2H), 4.40 (1H), 2.60 (2H), 2.22 (2H).

Example 9

D-benzylglutamate NCA

By using the same method and reaction scale of Example 8 andsubstituting H-d-Glu(OBn)-OH as starting material, reaction withphosgene for 1.25 hours at 50° C. afforded 75.53 g (Yield=95.6%) ofd-Glu(OBn) NCA as a colorless, crystalline solid. ¹H NMR (CDCl₃):identical to Example 8.

Example 10

mPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(Tyr(OBn)₃₀-co-d-Phe₁₀)-Ac

m-PEG12k-NH₂, (119.7 g, 10.0 mmol) was weighed into an oven-dried, 2L-round-bottom flask, dissolved in toluene (1 L), and dried byazeotropic distillation. After distillation to dryness, the polymer wasleft under vacuum for three hours. The flask was subsequently backfilledwith N₂, re-evacuated under reduced pressure, and dryN-methylpyrrolidone (NMP) (1100 mL) was introduced by cannula. Themixture was briefly heated to 40° C. to expedite dissolution and thenrecooled to 25° C. Glu(OBn) NCA (13.16 g, 50.0 mmol) and d-Glu(OBn) NCA(13.16 g, 50.0 mmol) were added to the flask, and the reaction mixturewas allowed to stir for 16 hours at ambient room temperature undernitrogen gas. Then, d-Phe NCA (19.12 g, 100 mmol) and Tyr (OBn) NCA(89.19 g, 300 mmol) were added and the solution was allowed to stir at35° C. for 48 hours at which point the reaction was complete (GPC,DMF/0.1% LiBr). The solution was cooled to room temperature and aceticanhydride (10.21 g, 100 mmol, 9.45 mL), N-methylmorpholine (NMM) (11.13g, 110 mmol, 12.1 mL) and dimethylaminopyridine (DMAP) (1.22 g, 10.0mmole) were added. Stirring was continued for 1 day at room temperature.The polymer was precipitated into diethyl ether (14 L) and isolated byfiltration, washed with fresh 500 mL portions of diethyl ether, anddried in vacuo to give the block copolymer as a fine, nearly colorlesspowder (214.7 g, Yield=92.3%). ¹H NMR (d₆-DMSO) δ 8.42-7.70 (theo. 50H,obs'd. 47H), 7.30 (theo. 250H, obs'd. 253H), 6.95 (theo. 120H, obs'd.122H), 5.10-4.85 (theo. 80H, obs'd. 80H), 4.65-4.20 ((theo. 50H, obs'd.56H), 3.72-3.25 (theo. 1087H, obs'd. 1593H), 3.05-2.45 (theo. 80H,obs'd. 83H), 2.44-1.60 (theo. 40H, obs'd. 42H).

Example 11

mPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly(Tyr(OH)₃₀-co-d-Phe₁₀)-Ac

mPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(Tyr(OBn)₃₀-co-d-Phe₁₀)-Ac from Example 10 (151.3 g, 6.5 mmol) andpentamethylbenzene (86.1 g, 0.58 mole) were dissolved into 1400 mL oftrifluoroacetic acid (TFA). The reaction was rapidly stirred for sixhours at room temperature. The TFA was removed on a rotary evaporatorwith the water bath temperature not exceeding 35° C. The resultant stiffpaste was dissolved in 800 mL of dry THF and the crude product wasprecipitated into 12 L diethyl ether while cooling to −30° C. Theresultant solid was collected by filtration, redissolved in 500 mL ofdry THF and reprecipitated into 3 L diethyl ether. A nearly colorless,odorless, fluffy polymer was obtained after drying the product overnightin vacuo (126.0 g, Yield=94.2%). ¹H NMR (d₆-DMSO) δ 9.09 (theo. 30H,obs'd. 29.4H), 8.50-7.75 (theo. 50H, obs'd. 52.7H), 7.40-6.45 (theo.220H, obs'd. 220H), 5.04 (theo. 20H, obs'd. 17.5H), 4.70-4.20 (theo.50H, obs'd. 54.5H), 3.91-3.05 (theo. 1087H, obs'd. 1391H), 3.03-2.10(theo. 80H, obs'd. 91H), 2.09-1.50 (theo. 40H, obs'd. 46H).

Example 12

mPEG12K-b-Poly-[d-Glu(NHOH)₅-co-Glu(NHOH)₅]-b-Poly(Tyr(OH)₃₀-co-d-Phe₁₀)-Ac

mPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly(Tyr(OH)₃₀-co-d-Phe₁₀)-Ac(113.3 g, 5.5 mmol) was dissolved in 1130 mL of dry THF and treated withhydroxylamine solution (50% aqueous, 2.20 mole, 146 mL) and1,5,7-triazabicyclo [4.4.0]dec-5-ene (TBD, 2.30 g, 16.5 mmol). Theresultant slightly turbid solution was stirred at 50° C. for 19 hoursunder N₂, cooled to room temperature and diluted with 1130 mL MeOH. Thecrude product was precipitated from 8 L diethyl ether while cooling to−30° C. The resultant solid was collected by filtration, redissolved ina mixture of 250 mL of dry THF and 125 mL acetone, treated with aceticacid (4.72 g, 79 mmol, 4.5 mL), heated to reflux for five minutes, andthen allowed to stir at ambient temperature for 1.5 hours. The productwas precipitated by addition of 2 L diethyl ether, collected by suctionfiltration, washed with fresh portions of diethyl ether, and driedovernight in vacuo to afford 106.3 g (Yield=97.5%) of nearly colorless,fluffy polymer. ¹H NMR (d₆-DMSO δ 9.12 (theo. 30H, obs'd. 30H),8.80-7.75 (theo. 50H, obs'd. 38.4H), 7.15 (theo. 50H, obs'd. 50H), 6.80(theo. 120H, obs'd. 120H), 4.65-4.05 (theo. 50H, obs'd. 50.4H),3.80-3.15 (theo. 1087H, obs'd. 1360H), 3.00-2.20 (theo. 80H, obs'd.79H), 2.15-1.60 (theo. 40H, obs'd. 40H).

Example 13

mPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(d-Leu₅-co-Asp(OtBu)₁₀-co-Tyr(OBn)₂₅)-Ac

Using the general protocol detailed in Example 10 and substituting theappropriate NCA starting materials afforded a crude polymer that wasprecipitated with 12 volumes of diethyl ether, then reprecipitated fromdichloromethane/diethyl ether: 1,12. After filtration and drying invacuo, the title compound (Yield=93.9%) was obtained as a fine,colorless, odorless solid.

Example 14

mPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(d-Leu₅-co-Asp(OH)₁₀-co-Tyr(OH)₂₅)-Ac

By using the method of Example 11 and substitutingmPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(d-Leu₅-co-Asp(OtBu)₁₀-co-Tyr(OBn)₂₅)-Acas starting material, reaction for three hours, 15 minutes at roomtemperature afforded the title product (Yield=97.0%) as a fluffy,colorless, odorless polymer.

Example 15

mPEG12K-b-Poly-(d-Glu(NHOH)₅-co-Glu(NHOH)₅)-b-Poly(d-Leu₅-co-Asp(OH)₁₀-co-Tyr(OH)₂₅)-Ac

mPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(d-Leu₅-co-Asp(OH)₁₀-co-Tyr(OH)₂₅)-Ac(20.81 g, 1.0 mmol) was dissolved in 210 mL of THF and treated withhydroxylamine solution (50% aqueous, 0.80 mole, 53.0 mL) and1,5,7-triazabicyclo [4.4.0]dec-5-ene (TBD, 0.84 g, 6.0 mmol). Theresultant slightly turbid solution was stirred at 50° C. for 17 hoursunder N₂, cooled to room temperature and diluted with 210 mL of MeOH.The crude product was precipitated with 1 L diethyl ether, filtered,washed with fresh portions of diethyl ether, and dried overnight invacuo (Yield=93.3%, hydroxylamine salt) as a fine, colorless polymer.

Example 16

mPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(d-Leu₃₀-co-Asp(OtBu)₁₀)-Ac

Using the general protocol detailed in Example 10 and substituting theappropriate NCA starting materials afforded a crude polymer that wasprecipitated with 30 volumes of diethyl ether/heptane: 6,1, thenreprecipitated from dichloromethane/diethyl ether: 1,20. Afterfiltration and drying in vacuo, the title compound (Yield=90.7%) wasobtained as a cream colored, odorless solid.

Example 17

mPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(d-Leu₃₀-co-Asp(OH)₁₀)-Ac

By using the method of Example 11, substitutingmPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(d-Leu₃₀-co-Asp(OtBu)₁₀)-Acas starting material and omitting PMB, reaction for two hours at roomtemperature afforded the title product (Yield=97.4%) as a fluffy,colorless polymer.

Example 18

mPEG12K-b-Poly-(d-Glu(NHOH)₅-co-Glu(NHOH)₅)-b-Poly(d-Leu₃₀-co-Asp(OH)₁₀)-Ac

By using the method of Example 12 and substitutingmPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(d-Leu₃₀-co-Asp(OH)₁₀)-Acas starting material, reaction for 17 hours at 50° C. afforded the titleproduct (Yield=96.4%, hydroxylamine salt) as a fine, colorless polymer.

Example 19

mPEG12K-b-Poly-(Glu(OBn)₁₀)-b-Poly(d-Phe₂₀-co-Tyr(OBn)₂₀)-Ac

Using the general protocol detailed in Example 10 and substituting theappropriate NCA starting materials afforded a crude polymer that wasprecipitated with 9 volumes of diethyl ether, then reprecipitated fromdichloromethane/diethyl ether: 1,14. After filtration and drying invacuo, the title compound (Yield=89%) was obtained as a cream colored,odorless solid.

Example 20

mPEG12K-b-Poly-(Glu(OBn)₁₀)-b-Poly(d-Phe₂₀-co-Tyr₂₀)-Ac

By using the method of Example 11, substitutingmPEG12K-b-Poly-(Glu(OBn)₁₀)-b-Poly(d-Phe₂₀-co-Tyr(OBz)₂₀)-Ac as startingmaterial and reacting for four hours at room temperature afforded thetitle product (Yield=87%) as a fluffy, colorless polymer.

Example 21

mPEG12K-b-Poly-(Glu(NHOH)₁₀)-b-Poly(d-Phe₂₀-co-Tyr₂₀)-Ac

By using the method of Example 12 and substitutingmPEG12K-b-Poly-(Glu(OBn)₁₀)-b-Poly(d-Phe₂₀-co-Tyr₂₀)-Ac as startingmaterial, reaction for 17 hours at 50° C. afforded the title product(Yield=94%, hydroxylamine salt) as a fine, colorless polymer.

Example 22

Synthesis of mPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(Tyr(OBn)₃₀-co-d-Phe₁₀)-Ac

m-PEG10k-NH₂ (119.7 g, 10.0 mmol, Example 3) was weighed into anoven-dried, 2 L-round-bottom flask, dissolved in toluene (1 L), anddried by azeotropic distillation. After distillation to dryness, thepolymer was left under vacuum for three hours. The flask wassubsequently backfilled with N₂, re-evacuated under reduced pressure,and dry N-methylpyrrolidone (NMP) (1100 mL) was introduced by cannula.The mixture was briefly heated to 40° C. to expedite dissolution andthen recooled to 25° C. Glu(OBn) NCA (13.16 g, 50.0 mmol) and d-Glu(OBn)NCA (13.16 g, 50.0 mmol) were added to the flask, and the reactionmixture was allowed to stir for 16 hours at ambient room temperatureunder nitrogen gas. Then, d-Phe NCA (19.12 g, 100 mmol) and Tyr (OBn)NCA (89.19 g, 300 mmol) were added and the solution was allowed to stirat 35° C. for 48 hours at which point the reaction was complete (GPC,DMF/0.1% LiBr). The solution was cooled to room temperature and aceticanhydride (10.21 g, 100 mmol, 9.45 mL), N-methylmorpholine (NMM) (11.13g, 110 mmol, 12.1 mL) and dimethylaminopyridine (DMAP) (1.22 g, 10.0mmole) were added. Stirring was continued for 1 day at room temperature.The polymer was precipitated into diethyl ether (14 L) and isolated byfiltration, washed with fresh 500 mL portions of diethyl ether, anddried in vacuo to give the block copolymer as a fine, nearly colorlesspowder (214.7 g, Yield=92.3%). ¹H NMR (d₆-DMSO) δ 8.42-7.70 (theo. 50H,obs'd. 47H), 7.30 (theo. 250H, obs'd. 253H), 6.95 (theo. 120H, obs'd.122H), 5.10-4.85 (theo. 80H, obs'd. 80H), 4.65-4.20 ((theo. 50H, obs'd.56H), 3.72-3.25 (theo. 1087H, obs'd. 1593H), 3.05-2.45 (theo. 80H,obs'd. 83H), 2.44-1.60 (theo. 40H, obs'd. 42H).

Example 23

Synthesis ofmPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OH)₃₀-co-d-Phe₁₀)-Ac

mPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OBn)₃₀-co-d-Phe₁₀)-Ac(151.3 g, 6.5 mmol) and pentamethylbenzene (86.1 g, 0.58 mole) weredissolved into 1400 mL of trifluoroacetic acid (TFA). The reaction wasrapidly stirred for six hours at room temperature. The TFA was removedon a rotary evaporator with the water bath temperature not exceeding 35°C. The resultant stiff paste was dissolved in 800 mL of dry THF and thecrude product was precipitated into 12 L diethyl ether while cooling to−30° C. The resultant solid was collected by filtration, redissolved in500 mL of dry THF and reprecipitated into 3 L diethyl ether. A nearlycolorless, odorless, fluffy polymer was obtained after drying theproduct overnight in vacuo (126.0 g, Yield=94.2%). ¹H NMR (d₆-DMSO) δ9.09 (theo. 30H, obs'd. 29.4H), 8.50-7.75 (theo. 50H, obs'd. 52.7H),7.40-6.45 (theo. 220H, obs'd. 220H), 5.04 (theo. 20H, obs'd. 17.5H),4.70-4.20 (theo. 50H, obs'd. 54.5H), 3.91-3.05 (theo. 1087H, obs'd.1391H), 3.03-2.10 (theo. 80H, obs'd. 91H), 2.09-1.50 (theo. 40H, obs'd.46H).

Example 24

Synthesis ofmPEG12K-b-Poly-[d-Glu(NHOH)₅-co-Glu(NHOH)₅]-b-Poly-(Tyr(OH)₃₀-co-d-Phe₁₀)-Ac

mPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OH)₃₀-co-d-Phe₁₀)-Ac(113.3 g, 5.5 mmol) was dissolved in 1130 mL of dry THF and treated withhydroxylamine solution (50% aqueous, 2.20 mole, 146 mL) and1,5,7-triazabicyclo [4.4.0]dec-5-ene (TBD, 2.30 g, 16.5 mmol). Theresultant slightly turbid solution was stirred at 50° C. for 19 hoursunder N₂, cooled to room temperature and diluted with 1130 mL MeOH. Thecrude product was precipitated from 8 L diethyl ether while cooling to−30° C. The resultant solid was collected by filtration, redissolved ina mixture of 250 mL of dry THF and 125 mL acetone, treated with aceticacid (4.72 g, 79 mmol, 4.5 mL), heated to reflux for five minutes, andthen allowed to stir at ambient temperature for 1.5 hours. The productwas precipitated by addition of 2 L diethyl ether, collected by suctionfiltration, washed with fresh portions of diethyl ether, and driedovernight in vacuo to afford 106.3 g (Yield=97.5%) of nearly colorless,fluffy polymer. ¹H NMR (d₆-DMSO δ 9.12 (theo. 30H, obs'd. 30H),8.80-7.75 (theo. 50H, obs'd. 38.4H), 7.15 (theo. 50H, obs'd. 50H), 6.80(theo. 120H, obs'd. 120H), 4.65-4.05 (theo. 50H, obs'd. 50.4H),3.80-3.15 (theo. 1087H, obs'd. 1360H), 3.00-2.20 (theo. 80H, obs'd.79H), 2.15-1.60 (theo. 40H, obs'd. 40H).

Example 25

Synthesis ofmPEG12K-b-Poly-(Asp(OtBu)₁₀-b-Poly-(Tyr(OBn)₂₀-co-d-Glu(OBn)₂₀-Ac

Using the protocol detailed in Example 22, replacing the NMP solventwith dichloromethane: DMF: 10,1, and substituting the appropriate NCAstarting materials, the title compound (Yield=93.9%) was prepared as afine, colorless, odorless solid. ¹H NMR (d₆-DMSO) δ 8.42-7.85 (theo.50H, obs'd. 51H), 7.30 (theo. 200H, obs'd.198H), 6.98 (theo. 80H, obs'd.72H), 5.15-4.85 (theo. 80H, obs'd. 80H), 4.68-4.20 (theo. 50H, obs'd.46H), 3.72-3.25 (theo. 1087H, obs'd. 1415H), 3.05-1.50 (theo. 120H,obs'd. 114H), 1.35 (theo. 90H, obs'd. 76H).

Example 26

Synthesis ofmPEG12K-b-Poly-(Asp(OH)₁₀-b-Poly-(Tyr(OH)₂₀-co-d-Glu(OBn)₂₀-Ac

By using the method of Example 23 and substitutingmPEG12K-b-Poly-(Asp(OtBu)₁₀-b-Poly-(Tyr(OBn)₂₀-co-d-Glu(OBn)₂₀-Ac asstarting material, reaction for three hours, 15 minutes at roomtemperature and precipitation from a mixture of dichloromethane, diethylether: 1,8.5 afforded the title product (Yield=98.9%) as a fine,colorless, odorless polymer. ¹H NMR (d₆-DMSO) δ 12.38 (theo. 10H, obs'd.9H), 9.13 (theo. 20H, obs'd. 17H), 8.40-7.80 (theo. 50H, obs'd. 43H),7.32 (theo. 100H, obs'd. 82H), 6.80 (theo. 80H, obs'd. 83H), 5.04 (theo.40H, obs'd. 34.2H), 4.60-4.20 (theo. 50H, obs'd. 55H), 3.80-3.20 (theo.1087H, obs'd. 1100H), 2.95-1.45 (theo. 140H, obs'd. 154.6H)

Example 27

Synthesis ofmPEG12K-b-Poly-(Asp(OH)₁₀-b-Poly-(Tyr(OH)₂₀-co-d-Glu(NHOH)₂₀-Ac

mPEG12K-b-Poly-(Asp(OH)₁₀-b-Poly-(Tyr(OH)₂₀-co-d-Glu(OBn)₂₀-Ac (20.81 g,1.0 mmol) was dissolved in 210 mL of THF and treated with hydroxylaminesolution (50% aqueous, 0.80 mole, 53.0 mL) and 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD, 0.84 g, 6.0 mmol). The resultant slightly turbidsolution was stirred at 50° C. for 17 hours under N₂, cooled to roomtemperature and diluted with 210 mL of MeOH. The crude product wasprecipitated with 1 L diethyl ether, filtered, washed with freshportions of diethyl ether, and dried overnight in vacuo to afford 19.68g (Yield=98.5%) of colorless, fluffy polymer as the hydroxylamine salt.A portion of the hydroxylamine salt (10.0 g) was dissolved in 1 L of 30%tert-butyl alcohol/water, treated with ammonium carbonate (3.33 g), andlyophilized to afford the native carboxylic acid salt form (quantitativeyield) as a colorless, odorless, fluffy solid. ¹H NMR (d₆-DMSO,hydroxylamine salt) δ 9.08 (theo. 20H, obs'd. 10H), 6.80 (theo. 80H,obs'd. 80H), 4.60-4.02 (theo. 50H, obs'd. 54.7H), 3.80-3.15 (theo.1087H, obs'd. 1211H), 2.90 (theo. 40H, obs'd. 45H), 2.80-1.50 (theo.100H, obs'd. 120H). Spectrum showed traces of solvent that affectedintegration in the upheld region.

Example 28

Synthesis ofmPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(d-Leu₅-co-Asp(OtBu)₁₀-co-Tyr(OBn)₂₅)-Ac

Using the general protocol detailed in Example 22 and substituting theappropriate NCA starting materials afforded a crude polymer that wasprecipitated with 12 volumes of diethyl ether, then reprecipitated fromdichloromethane/diethyl ether: 1,12. After filtration and drying invacuo, the title compound (Yield=89.2%) was obtained as a fine,colorless, odorless solid. ¹H NMR (d₆-DMSO) δ 8.52-7.75 (theo. 50H,obs'd. 49H), 7.35 (theo. 175H, obs'd. 198H), 7.11 (theo. 50H, obs'd.49H), 6.80 (theo. 50H, obs'd. 50H), 5.10-4.75 (theo. 70H, obs'd. 75H),4.70-4.15 (theo. 50H, obs'd. 56H), 3.72-3.25 (theo. 1087H, obs'd.1580H), 3.05-1.65 (theo. 110H, obs'd. 144H), 1.58-0.55 (theo. 135H,obs'd. 155H).

Example 29

Synthesis ofmPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(d-Leu₅-co-Asp(OH)₁₀-co-Tyr(OH)₂₅)-Ac

By using the method of Example 23 and substitutingmPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(d-Leu₅-co-Asp(OtBu)₁₀-co-Tyr(OBn)₂₅)-Acas starting material, reaction for three hours, 15 minutes at roomtemperature and precipitations from a mixture of dichloromethane,diethyl ether: 1,24 followed by dichloromethane, diethyl ether: 1,12afforded the title product (Yield=97.0%) as a fluffy, colorless,odorless polymer. ¹H NMR (d₆-DMSO)) δ 9.4-8.5 (theo. 35H, obs'd. 34H),8.40-7.75 (theo. 50H, obs'd. 61H), 7.35-7.15 (theo. 50H, obs'd. 43H),6.98 (theo. 50H, obs'd. 49H), 6.60 (theo. 50H, obs'd. 50H), 5.04 (theo.20H, obs'd. 18H), 4.65-4.10 (theo. 50H, obs'd. 58H), 3.80-3.20 (theo.1087H, obs'd. 1367H, contains masked H₂O peak), 3.00-2.15 (theo. 90H,obs'd. 95H), 2.05-1.70 (theo. 20H, obs'd. 26H), 1.63-0.57 (theo. 45H,obs'd. 45H).

Example 30

Synthesis ofmPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(d-Leu₅-co-Asp(OH)₁₀-co-Tyr(OH)₂₅)-Ac

By using the method of Example 27 and substitutingmPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(d-Leu₅-co-Asp(OH)₁₀-co-Tyr(OH)₂₅)-Acas starting material, reaction for 12 hours at 50° C. afforded the titleproduct (Yield=93.3%, hydroxylamine salt) as a fine, colorless polymer.¹H NMR (d₆-DMSO) δ 9.4-8.5 (theo. 35H, obs'd. 34H), 8.60-7.75 (theo.50H, obs'd. 43H), 7.2-6.85 (theo. 50H, obs'd. 55H), 6.60 (theo. 50H,obs'd. 50H), 4.60-4.00 (theo. 50H, obs'd. 41H), 3.80-3.00 (theo. 1087H,obs'd. 1174H, contains masked H₂O peak), 3.00-1.65 (theo. 110H, obs'd.124H), 1.63-0.57 (theo. 45H, obs'd. 40H).

Example 31

Synthesis ofmPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(d-Leu₃₀-co-Asp(OtBu)₁₀)-Ac

Using the general protocol detailed in Example 22 and substituting theappropriate NCA starting materials afforded a crude polymer that wasprecipitated with 30 volumes of diethyl ether/heptane: 6,1, thenreprecipitated from dichloromethane/diethyl ether: 1,20. Afterfiltration and drying in vacuo, the title compound (Yield=90.7%) wasobtained as a cream colored, odorless solid. ¹H NMR (d₄₋MeOH) δ 7.31(theo. 50H, obs'd. 66H), 5.04 (theo. 20H, obs'd. 20H), 4.45-3.97 (theo.50H, obs'd. 37H), 3.95-3.25 (theo. 1087H, obs'd. 1876H), 3.05-0.80(theo. 420H, obs'd. 313H).

Example 32

Synthesis ofmPEG12K-b-Poly-(d-Glu(NHOH)₅-co-Glu(NHOH)₅)-b-Poly(d-Leu₅-co-Asp(OH)₁₀-co-Tyr(OH)₂₅)-Ac

By using the method of Example 27 and substitutingmPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(d-Leu₅-co-Asp(OH)₁₀-co-Tyr(OH)₂₅)-Acas starting material, reaction for 12 hours at 50° C. afforded the titleproduct (Yield=93.3%, hydroxylamine salt) as a fine, colorless polymer.¹H NMR (d₆-DMSO) δ 9.4-8.5 (theo. 35H, obs'd. 34H), 8.60-7.75 (theo.50H, obs'd. 43H), 7.2-6.85 (theo. 50H, obs'd. 55H), 6.60 (theo. 50H,obs'd. 50H), 4.60-4.00 (theo. 50H, obs'd. 41H), 3.80-3.00 (theo. 1087H,obs'd. 1174H, contains masked H₂O peak), 3.00-1.65 (theo. 110H, obs'd.124H), 1.63-0.57 (theo. 45H, obs'd. 40H).

Example 33

Synthesis ofmPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(d-Leu₃₀-co-Asp(OH)₁₀)-Ac

By using the method of Example 23, substitutingmPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(d-Leu₃₀-co-Asp(OtBu)₁₀)-Acas starting material and omitting PMB, reaction for two hours at roomtemperature and precipitation from dichloromethane, diethyl ether: 1, 13afforded the title product (Yield=97.4%) as a fluffy, colorless polymer.¹H NMR (d₄₋MeOH) δ 7.31 (theo. 50H, obs'd. 61H), 5.04 (theo. 20H, obs'd.20H), 4.45-3.97 (theo. 50H, obs'd. 29H), 3.95-3.25 (theo. 1087H, obs'd.1542H), 3.05-0.80 (theo. 330H, obs'd. 258H).

Example 34

Synthesis ofmPEG12K-b-Poly-(d-Glu(NHOH)₅-co-Glu(NHOH)₅)-b-Poly(d-Leu₃₀-co-Asp(OH)₁₀)-Ac

By using the method of Example 27 and substitutingmPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(d-Leu₃₀-co-Asp(OH)₁₀)-Acas starting material, reaction for 17 hours at 50° C. afforded the titleproduct (Yield=96.4%, hydroxylamine salt) as a fine, colorless polymer.¹H NMR (d₆-DMSO) δ 8.8-7.2 (theo. 70H, obs'd. 67H), 4.55-3.85 (theo.50H, obs'd. 50H), 3.80-3.30 (theo. 1087H, obs'd. 1520H), 3.29-2.60(theo. 60H, obs'd. 80H),

2.42-0.70 (theo. 270H, obs'd. 278H).

Example 35

Synthesis ofmPEG12K-b-Poly-[d-Glu(NHOH)₅-co-Glu(NHOH)₅]-b-Poly-(Tyr(OH)₃₀-co-d-Phe₁₀)-Ac

mPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OH)₃₀-co-d-Phe₁₀)-Ac(30.86 g, 1.50 mmol) was dissolved in 310 mL of dry THF and treated withhydroxylamine solution (50% aqueous, 0.60 mole, 39.7 mL) and1,5,7-triazabicyclo [4.4.0]dec-5-ene (TBD, 626.4 mg, 4.5 mmol). Theresultant slightly turbid solution was stirred at room temperature for69 hours under N₂ and diluted with 310 mL MeOH. The crude product wasprecipitated from 3 L diethyl ether while cooling to −30° C. Theresultant solid was collected by filtration, redissolved in a mixture of150 mL of dry THF and 100 mL acetone, treated with acetic acid (1.26 g,21.0 mmol, 1.2 mL) and then allowed to stir at ambient temperature for 2hours. The product was precipitated by addition of 1.5 L diethyl ether,collected by suction filtration, washed with fresh portions of diethylether, and dried overnight in vacuo to afford 29.41 g (Yield=98.9%) ofnearly colorless, fluffy polymer. ¹H NMR (d₆-DMSO): identical to Example24.

Example 36

Synthesis ofmPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly-(Tyr(OBn)₂₅-co-d-Phe₁₅)-Ac

Using the protocol detailed in Example 24 and substituting theappropriate NCA starting materials afforded a crude polymer that wasprecipitated with 10 volumes of diethyl ether. After filtration anddrying in vacuo, the title compound (Yield=83.6%) was obtained as afine, colorless, odorless solid. ¹H NMR (d₆-DMSO) δ 8.42-7.80 (theo.50H, obs'd. 43H), 7.42-6.68 (theo. 350H, obs'd. 350H), 5.10-4.80 (theo.70H, obs'd. 73H), 4.65-4.20 (theo. 50H, obs'd. 50H), 3.75-3.25 (theo.1087H, obs'd. 1755H), 3.01-2.30 (theo. 80H, obs'd. 85H), 2.02-1.60(theo. 40H, obs'd. 38H).

Example 37

Synthesis ofmPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

By using the method of Example 23 and substitutingmPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OBn)₂₅-co-d-Phe₁₅)-Acas starting material, reaction for 5.25 hours at room temperatureafforded the title product (Yield=99.3%) as a fine, colorless, odorlesspolymer. ¹H NMR (d₆-DMSO) δ 9.09 (theo. 25H, obs'd. 22H), 8.40-7.75(theo. 50H, obs'd. 49H), 7.40-6.50 (theo. 225H, obs'd. 225H), 5.04(theo. 20H, obs'd. 21H), 4.65-4.20 (theo. 50H, obs'd. 54H), 3.81-3.20(theo. 1087H, obs'd. 1613H), 3.05-2.10 (theo. 80H, obs'd. 90H),2.05-1.63 (theo. 40H, obs'd. 38H).

Example 38

Synthesis ofmPEG12K-b-Poly-[d-Glu(NHOH)₅-co-Glu(NHOH)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

mPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac(51.23 g, 2.50 mmol) was dissolved in 515 mL of dry THF and treated withhydroxylamine solution (50% aqueous, 1.00 mole, 66.3 mL, 40 equiv./Bnester moiety) and 1,5,7-triazabicyclo [4.4.0]dec-5-ene (TBD, 1.044 g,7.5 mmol, 0.3 equiv.). The resultant slightly turbid solution wasstirred at room temperature for 108 hours under N₂ and diluted with 515mL of IPA. The crude product was precipitated from 6 L of diethyl ether.The resultant solid was collected by filtration, redissolved in amixture of 300 mL of dry THF and 200 mL acetone, treated with aceticacid (2.25 g, 37.5 mmol, 2.15 mL), and then allowed to stir at ambienttemperature for 2.5 hours. The product was precipitated by addition of 3L of diethyl ether, collected by suction filtration, washed with freshportions of diethyl ether, and dried overnight in vacuo to afford 45.16g (Yield=91.5%) of the title compound as a nearly colorless, fluffypolymer with a slight odor of acetic acid. ¹H NMR (d₆-DMSO) δ 9.35-8.85(theo. 45H, obs'd. 28H), 8.42-7.75 (theo. 50H, obs'd. 37H), 7.37-6.46(theo. 175H, obs'd. 164H), 4.65-4.00 (theo. 50H, obs'd. 50H), 3.82-3.07(theo. 1087H, obs'd. 1708H, contains masked H₂O peak), 3.05-2.20 (theo.80H, obs'd. 84H), 2.18-1.63 (theo. 40H, obs'd. 68H, contains trace ofHOAc).

Example 39

Synthesis ofmPEG12K-b-Poly-[d-Glu(NHOH)₅-co-Glu(NHOH)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

By using the method of Example 38 and increasing the hydroxylamineconcentration (80 equiv./Bn ester), reaction for 65 hours at roomtemperature and work-up as above afforded the title product(Yield=87.8%) as a fine, colorless polymer with a slight odor of aceticacid. ¹H NMR (d₆-DMSO): identical to Example 38.

Example 40

Synthesis ofmPEG12K-b-Poly-[d-Glu(NHOH)₅-co-Glu(NHOH)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

By using the method of Example 39 and substitution of TBD with2-hydroxypyridine (0.3 equiv.), reaction for 137 hours at roomtemperature and work-up as above afforded the title product(Yield=91.2%) as a fine, colorless polymer with a slight odor of aceticacid. ¹H NMR (d₆-DMSO): identical to Example 38.

Example 41

Synthesis ofmPEG12K-b-Poly-[d-Glu(NHOH)₅-co-Glu(NHOH)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

By using the method of Example 38 and substitution of TBD with2-hydroxypyridine (0.3 equiv.), reaction at 50° C. for 24.5 hours andwork-up as above afforded the title product (Yield=91.2%) as a fine,colorless polymer with a slight odor of acetic acid. ¹H NMR (d₆-DMSO):identical to Example 38.

Example 42

Synthesis ofmPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly-(Tyr(OBn)₂₅-co-d-Phe₁₅)-Ac

Using the protocol detailed in Example 36 and substituting theappropriate NCA starting materials afforded a crude polymer that wasprecipitated with 5 volumes of isopropanol. After filtration and dryingin vacuo, the title compound (Yield=84.2%) was obtained as a fine,colorless, odorless solid. ¹H NMR (d₆-DMSO): identical to Example 36.

Example 43

Synthesis ofmPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

By using the method of Example 37, reaction ofmPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OBn)₂₅-co-d-Phe₁₅)-Acwith PMB in TFA for four hours at room temperature and precipitationfrom a mixture of chlorobutane, TBME: 1,3 afforded the title product(Yield=93.1%) as a fine, colorless, odorless polymer. ¹H NMR (d₆-DMSO):identical to Example 37.

Example 44

Synthesis ofmPEG12K-b-Poly-[d-Glu(NHOH)₅-co-Glu(NHOH)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

mPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac(4.10 g, 0.20 mmol) was dissolved in 41 mL of dry THF and treated withhydroxylamine solution (50% aqueous, 40.0 mmol, 2.65 mL, 20 equiv./Bnester moiety) and lithium hydroxide monohydrate (84.0 mg, 2.0 mmol, 1.0equiv./Bn ester moiety). The resultant clear pale yellow solution wasstirred at room temperature for 22 hours under N₂ and diluted with 41 mLof IPA. The crude product was precipitated from 160 mL of TBME withrapid stirring. The resultant solid was collected by filtration, driedin vacuo, and redissolved in a mixture of 24 mL dry THF and 16 mLacetone. The solution was treated with acetic acid (0.18 g, 3.00 mmol,0.17 mL), briefly heated to reflux, and allowed to stir at ambienttemperature for 15 hours. The product was precipitated by addition ofvolumes of TBME, collected by suction filtration, washed with freshportions of TBME, and dried overnight in vacuo to afford 3.62 g(Yield=91.7%) of the title compound as a nearly colorless, fluffypolymer. ¹H NMR (d₆-DMSO): identical to Example 38.

Example 45

Synthesis ofmPEG12K-b-Poly-[d-Glu(NHOH)₅-co-Glu(NHOH)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

Using the method described above in Example 44,mPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Acwas converted to the title compound using lithium hydroxide monohydrate(2.0 equiv./Bn ester moiety). Reaction time was 18 hours. The crudeproduct was precipitated from 16 volumes of IPA and the resultant solidwas treated with THF, acetone, and acetic acid as detailed in Example44. After precipitation from two volumes of TBME, filtration, and dryingin vacuo, the title compound (Yield=96.2%) was obtained as a fine,colorless solid. ¹H NMR (d₆-DMSO): identical to Example 38.

Example 46

Synthesis ofmPEG12K-b-Poly-[d-Glu(NHOH)₅-co-Glu(NHOH)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

mPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac(2.05 g, 0.10 mmol) was dissolved in 21 mL of methanol and treated withhydroxylamine solution (50% aqueous, 20.0 mmol, 1.32 mL, 20 equiv./Bnester moiety) and 1M lithium hydroxide solution (1.0 mL, 1.0 mmol, 1.0equiv./Bn ester moiety). The resultant pale yellow solution was stirredat room temperature for 22 hours under N₂ and then an additional portionof 1M lithium hydroxide solution (1.0 mL, 1.0 mmol, 1.0 equiv./Bn estermoiety) was added. After an additional 24 hours, the crude product wasprecipitated from 160 mL of TBME. The resultant solid was collected byfiltration, dried in vacuo, and redissolved in a mixture of 12 mL dryTHF and 8 mL acetone. The solution was treated with acetic acid (0.21 g,3.50 mmol, 0.20 mL), briefly heated to reflux, and allowed to stir atambient temperature for 16 hours. The product was precipitated byaddition of 40 mL TBME, collected by suction filtration, washed withfresh portions of TBME, and dried overnight in vacuo to afford 1.87 g(Yield=94.9%) of the title compound as a nearly colorless, fluffypolymer. ¹H NMR (d₆-DMSO): identical to Example 38.

Example 47

Synthesis ofmPEG12K-b-Poly-[d-Glu(NHOH)₅-co-Glu(NHOH)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

Using the method described above in Example 44,mPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Acwas converted to the title compound using lithium hydroxide solution(0.5 equiv./Bn ester moiety). Reaction time was 72 hours. The solutionwas diluted with one volume of IPA, and crude product was precipitatedfrom two volumes of TBME. The resultant solid was treated with THF,acetone, and acetic acid as detailed in Example 44. After precipitationfrom two volumes of TBME, filtration, and drying in vacuo, the titlecompound (Yield=91.1%) was obtained as a fine, colorless solid. ¹H NMR(d₆-DMSO): identical to Example 38.

Example 48

Synthesis ofmPEG12K-b-Poly-[d-Glu(NHOH)₅-co-Glu(NHOH)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

Using the method described above in Example 47,mPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Acwas converted to the title compound using 1M potassium hydroxidesolution (2.0 equiv./Bn ester moiety). Reaction time was 6 hours. Workupafforded the title compound (Yield=92.4%) as a fine, colorless solid. ¹HNMR (d₆-DMSO): identical to Example 38.

Example 49

Synthesis of mPEG12K-b-Poly-(d-Glu(OBn)_(3.5)-co-Glu(OBn)_(3.5))-b-Poly(Tyr(OBn)₂₅-co-d-Phe₁₅)-Ac

m-PEG10k-NH₂, (59.86 g, 5.0 mmol) was weighed into an oven-dried, 1L-round-bottom flask, dissolved in toluene (450 mL), and dried byazeotropic distillation. After distillation to dryness, the polymer wasleft under vacuum for 16 hours. The flask was subsequently backfilledwith N₂, re-evacuated under reduced pressure, and dryN-methylpyrrolidone (NMP, 250 mL) and then dichloromethane (250 mL) wereintroduced by cannula. The mixture was briefly heated to 40° C. toexpedite dissolution and then recooled to 25° C. Glu(OBn) NCA (4.61 g,17.5 mmol) and d-Glu(OBn) NCA (4.61 g, 17.5 mmol) were added to theflask, and the reaction mixture was allowed to stir for 24 hours atambient room temperature under nitrogen gas. Then, d-Phe NCA (14.34 g,75.0 mmol) and Tyr (OBn) NCA (37.16 g, 125.0 mmol) were added and thesolution was allowed to stir at room temperature for three days and thenheated 35° C. for 7 hours at which point the reaction was complete (GPC,DMF/0.1% LiBr). The solution was cooled to room temperature and aceticanhydride (5.11 g, 50.0 mmol, 4.80 mL), N-methylmorpholine (NMM) (5.56g, 55.0 mmol, 6.1 mL) and dimethylaminopyridine (DMAP) (0.61 g, 5.0mmole) were added. Stirring was continued for 18 hours at roomtemperature and the dichloromethane was removed on the rotaryevaporator. The polymer was precipitated into isopropanol (2.6 L) andisolated by filtration, washed with fresh 500 mL portions ofisopropanol, and dried in vacuo to give the block copolymer as a fine,nearly colorless powder (102.40 g, Yield=92.6%). ¹H NMR (d₆-DMSO) δ8.42-7.80 (theo. 47H, obs'd. 44H), 7.35 (theo. 75H, obs'd. 75H),7.28-6.65 (theo. 125H, obs'd. 125H), 5.10-4.84 (theo. 64H, obs'd. 59H),4.64-4.20 (theo. 47H, obs'd. 39H), 3.72-3.25 (theo. 1087H, obs'd.16713H), 3.00-2.20 (theo. 80H, obs'd. 88H), 2.03-1.60 (theo. 28H, obs'd.27H).

Example 50

Synthesis of mPEG12K-b-Poly-(d-Glu(OBn)_(3.5)-co-Glu(OBn)_(3.5))-b-Poly(Tyr(OBn)₂₅-co-d-Phe₁₅)-Ac

Using the protocol detailed in Example 49 with dry N-methylpyrrolidone(NMP, 125 mL) and dichloromethane (375 mL) as solvents afforded a crudepolymer that was precipitated with 5 volumes of isopropanol. Afterfiltration and drying in vacuo, the title compound (Yield=96.5%) wasobtained as a fine, colorless, odorless solid. ¹H NMR (d₆-DMSO):identical to Example 49.

Example 51

Synthesis ofmPEG12K-b-Poly-[d-Glu(NHOH)₅-co-Glu(NHOH)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

mPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac(4.10 g, 0.20 mmol) was dissolved in 41 mL of THF and treated withhydroxylamine solution (2.65 mL, 40.0 mmol) and 1M potassium hydroxide(2.0 mL, 2.0 mmol, 1.0 equiv./Bn ester moiety). The resultant slightlyhazy pink solution was stirred at room temperature for 42 hours under N₂and then diluted with acetone (58.1 g, 1.0 mol, 74 mL). Acetic acid(2.40 g, 40.0 mmol, 2.3 mL) was added, the solution was briefly heatedto reflux, and then was stirred at room temperature for four hours. Theproduct was precipitated with TBME (300 mL) using vigorous stirring.After stirring an additional 30 minutes, filtration and drying in vacuoafforded the title compound (Yield=92.9%) as a fine, colorless solid. ¹HNMR (d₆-DMSO): identical to Example 38.

Example 52

Synthesis ofmPEG12K-b-Poly-[d-Glu(NHOH)₅-co-Glu(NHOH)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

Using the method described above in Example 51,mPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Acwas converted to the title compound using 1M lithium hydroxide solution(2.0 equiv./Bn ester moiety). Reaction time was 6 hours. Workup as aboveand dilution with IPA (1 volume) followed by precipitation with TBME (3volumes) afforded the title compound (Yield=90.6%) as a fine, colorlesssolid. ¹H NMR (d₆-DMSO): identical to Example 38.

Example 53

Synthesis ofmPEG12K-b-Poly-[d-Glu(NHOH)₅-co-Glu(NHOH)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

Using the method described above in Example 52,mPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Acwas converted to the title compound using solid lithium hydroxidemonohydrate (2.0 equiv./Bn ester moiety). Reaction time was 6 hours.Workup afforded the title compound (Yield=99.2%) as a fine, colorlesssolid. ¹H NMR (d₆-DMSO): identical to Example 38.

Example 54

Synthesis ofmPEG12K-b-Poly-[d-Glu(OBn)_(3.5)-co-Glu(OBn)_(3.5)]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

By using the method of Example 37, reaction ofmPEG12K-b-Poly-[d-Glu(OBn)_(3.5)-co-Glu(OBn)_(3.5)]-b-Poly-(Tyr(OBn)₂₅-co-d-Phe₁₅)-Acwith PMB in TFA for 3.5 hours at room temperature and precipitation froma mixture of dichloromethane, TBME: 1,7 afforded the title product(Yield=96.1%) as a fine, colorless, odorless polymer. ¹H NMR (d₆-DMSO) δ9.09 (theo. 25H, obs'd. 22H), 8.46-7.79 (theo. 47H, obs'd. 48H),7.40-6.45 (theo. 210H, obs'd. 229H), 5.04 (theo. 14H, obs'd. 13H),4.65-4.20 (theo. 47H, obs'd. 47H), 3.81-3.15 (theo. 1087H, obs'd.1308H), 3.03-2.10 (theo. 80H, obs'd. 78H), 2.06-1.62 (theo. 40H, obs'd.27H).

Example 55

Synthesis ofmPEG12K-b-Poly-[d-Glu(NHOH)₅-co-Glu(NHOH)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

Using the method described above in Example 51,mPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Acwas converted to the title compound using 4M sodium hydroxide solution(2.0 equiv./Bn ester moiety). Reaction time was 4 hours. The solutionwas diluted with acetone (0.30 volumes based on total reaction mixturevolume) and acetic acid (1.0 equiv./hydroxylamine) was added. After 14hours, the crude product was precipitated from three volumes of TBME,stirred for three days, and filtered. The filter cake was washed withTBME (50 mL), TBME, IPA:20,1 (50 mL) and dried in vacuo to afford thetitle compound (Yield=93.5%) as a fine, colorless solid with a slightodor of acetic acid. ¹H NMR (d₆-DMSO): identical to Example 38.

Example 56

Synthesis ofmPEG12K-b-Poly-[d-Glu(NHOH)_(3.5)-co-Glu(NHOH)_(3.5)]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

Using the method described above in Example 52,mPEG12K-b-Poly-[d-Glu(OBn)_(3.5)-co-Glu(OBn)_(3.5)]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Acwas converted to the title compound using solid lithium hydroxidemonohydrate (2.0 equiv./Bn ester moiety). Reaction time was 6 hours.Workup afforded the title compound (Yield=94.9%) as a fine, colorlesssolid with a slight odor of acetic acid. ¹H NMR (d₆-DMSO) δ 10.2-9.2(theo. 25H, obs'd. 19H), 8.52-7.90 (theo. 47H, obs'd. 38H), 7.40-6.49(theo. 175H, obs'd. 175H), 4.63-4.00 (theo. 47H, obs'd. 42H), 3.84-3.11(theo. 1087H, obs'd. 1496H, contains masked H₂O peak), 3.00-2.20 (theo.80H, obs'd. 78H), 2.16-1.60 (theo. 28H, obs'd. ˜26H, containsoverlapping HOAc peak at δ1.69).

Example 57

Synthesis ofmPEG12K-b-Poly-[d-Glu(NHOH)_(3.5)-co-Glu(NHOH)_(3.5)]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

Using the method described above in Example 52,mPEG12K-b-Poly-[d-Glu(OBn)_(3.5)-co-Glu(OBn)_(3.5)]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Acwas converted to the title compound using 10M sodium hydroxide solution(2.0 equiv./Bn ester moiety). Reaction time was 3 hours. Workup affordedthe title compound (Yield=85.8%) as a fine, colorless solid with aslight odor of acetic acid. ¹H NMR (d₆-DMSO): identical to Example 56.

Example 58

Synthesis ofmPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(d-Phe₁₅-co-Asp(OtBu)₅-co-Tyr(OBn)₂₀)-Ac

Using the method detailed in Example 49 with anhydrous dichloromethane(2 parts) and N,N-dimethylacetamide (DMAC, 1 part) as solvents andsubstituting the appropriate NCA building blocks afforded a crudepolymer that was precipitated with 5 volumes of isopropanol. Afterfiltration and drying in vacuo, the title compound (Yield=95.4%) wasobtained as a fine, colorless, odorless solid. ¹H NMR (d₆-DMSO) δ8.57-7.75 (theo. 50H, obs'd. 47H), 7.41-6.67 (theo. 305H, obs'd. 305H),5.10-4.85 (theo. 60H, obs'd. 59H), 4.70-4.18 (theo. 50H, obs'd. 49H),3.72-3.25 (theo. 1087H, obs'd. 1131H), 3.05-2.20 (theo. 80H, obs'd.100H), 2.05-1.58 (theo. 40H, obs'd. 25H), 1.38-1.20 (theo. 45H, obs'd.40H).

Example 59

mPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly-(d-Leu₁₅-co-Asp(OtBu)₅-co-Tyr(OBn)₂₀)-Ac

Using the method detailed in Example 58 and substituting the appropriateNCA building blocks afforded a crude polymer that was precipitated with5 volumes of isopropanol. After filtration and drying in vacuo, thetitle compound (Yield=95.5%) was obtained as a fine, colorless, odorlesssolid. ¹H NMR (d₆-DMSO) δ 8.45-7.78 (theo. 50H, obs'd. 47H), 7.45-6.67(theo. 230H, obs'd. 230H), 5.10-4.80 (theo. 60H, obs'd. 59H), 4.65-4.00(theo. 50H, obs'd. 52H), 3.70-3.25 (theo. 1087H, obs'd. 1196H),3.05-2.55 (theo. 40H, obs'd. 41H), 2.48-2.30 (theo. 40H, obs'd. 33H),2.05-1.71 (theo. 40H, obs'd. 25H), 1.69-1.02 (theo. 60H, obs'd. 65H),0.95-0.55 (theo. 90H, obs'd. 83H).

Example 60

Synthesis ofmPEG12K-b-Poly-[d-Glu(NHOH)_(3.5)-co-Glu(NHOH)_(3.5)]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

Using the method described above in Example 57,mPEG12K-b-Poly-[d-Glu(OBn)_(3.5)-co-Glu(OBn)_(3.5)]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Acwas converted to the title compound using 4M sodium hydroxide solution(2.0 equiv./Bn ester moiety). Reaction time was 16 hours. Workup withthree times the normal volume of IPA followed by precipitation with TBMEafforded the title compound (Yield=92.7%) as a fine, pale cream-colored,solid with a slight odor of acetic acid.

Example 61

Synthesis ofmPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly-(d-Tyr(OBn)₂₀-co-Tyr(OBn)₂₀)-Ac

Using the mixed reaction solvent method detailed in Example 58 andsubstituting the appropriate NCA building blocks afforded a crudepolymer that was precipitated with 9 volumes of isopropanol. Afterfiltration and drying in vacuo, the title compound (Yield=96.6%) wasobtained as a fine, colorless, odorless solid. ¹H NMR (d₆-DMSO) δ8.44-7.80 (theo. 50H, obs'd. 47H), 7.40-6.75 (theo. 410H, obs'd. 410H),5.11-4.84 (theo. 100H, obs'd. 94H), 4.60-4.20 (theo. 50H, obs'd. 52H),3.70-3.25 (theo. 1087H, obs'd. 1605H), 3.00-2.28 (theo. 80H, obs'd.95H), 2.03-1.60 (theo. 40H, obs'd. 31H).

Example 62

Synthesis ofmPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly-(d-Leu₁₅-co-Asp(OH)₅-co-Tyr(OH)₂₀)-Ac

By using the method of Example 54, reaction ofmPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly-(d-Leu₁₅-co-Asp(OtBu)₅-co-Tyr(OBn)₂₀)-Acwith PMB in TFA for 3.5 hours at room temperature and precipitation froma mixture of dichloromethane, TBME: 1,6 afforded the title product(Yield=95.5%) as a fine, colorless, odorless polymer. ¹H NMR (d₆-DMSO) δ9.15 (theo. 20H, obs'd. 18H), 8.43-7.60 (theo. 50H, obs'd. 47H),7.40-6.45 (theo. 130H, obs'd. 130H), 5.04 (theo. 20H, obs'd. 13H),4.65-4.00 (theo. 50H, obs'd. 48H), 3.85-3.15 (theo. 1087H, obs'd.1334H), 3.01-2.10 (theo. 80H, obs'd. 80H), 2.05-1.65 (theo. 40H, obs'd.42H), 1.63-0.55 (theo. 90H, obs'd. 75H).

Example 63

Synthesis ofmPEG12K-b-Poly-[d-Glu(NHOH)₅-co-Glu(NHOH)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

Using the method described above in Example 47,mPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Acwas converted to the title compound using solid potassium hydroxide (2.0equiv./Bn ester moiety) pre-dissolved in the hydroxylamine solution.Reaction time was 5.5 hours. Workup afforded the title compound(Yield=74.0%) as a fine, colorless solid with a slight odor of aceticacid. ¹H NMR (d₆-DMSO): identical to Example 38.

Example 64

Synthesis ofmPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly-(d-Tyr(OH)₂₀-co-Tyr(OH)₂₀)-Ac

By using the method of Example 54, reaction ofmPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly-(d-Tyr(OBn)₂₀-co-Tyr(OBn)₂₀)-Acwith PMB in TFA for 4.5 hours at room temperature and precipitation froma mixture of dichloromethane, TBME: 1,5 afforded the title product(Yield=97.7%) as a fine, colorless, odorless solid. ¹H NMR (d₆-DMSO) δ9.1 (theo. 40H, obs'd. 33H), 8.36-7.77 (theo. 50H, obs'd. 52H),7.40-6.45 (theo. 210H, obs'd. 234H), 5.04 (theo. 20H, obs'd. 17H),4.60-4.20 (theo. 50H, obs'd. 50H), 4.02-3.15 (theo. 1087H, obs'd. 1384H,contains obscured water peak), 3.00-2.10 (theo. 80H, obs'd. 78H),2.06-1.62 (theo. 40H, obs'd. 39H).

Example 65

Synthesis ofmPEG12K-b-Poly-(d-Glu(NHOH)₅-co-Glu(NHOH)₅)-b-Poly-(d-Leu₁₅-co-Asp(OH)₅-co-Tyr(OH)₂₀)-Ac

Using the method described above in Example 52,mPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly-(d-Leu₁₅-co-Asp(OH)₅-co-Tyr(OH)₂₀)-Acwas converted to the title compound using lithium hydroxide monohydrate(2.0 equiv./Bn ester moiety). Reaction time was 15 hours. Workupfollowed by precipitation with IPA, TBME afforded the title compound(Yield=quantitative) as a fine, colorless solid with a slight odor ofacetic acid. ¹H NMR (d₆-DMSO) δ 10.2-9.0 (theo. 40H, obs'd. 31H),8.65-7.75 (theo. 50H, obs'd. 37H), 7.27-6.50 (theo. 80H, obs'd. 80H),4.61-4.00 (theo. 50H, obs'd. 58H), 3.90-3.15 (theo. 1087H, obs'd.1356H), 3.02-2.20 (theo. 80H, obs'd. 100H), 2.40-1.70 (theo. 40H, obs'd.˜47H, contains overlapping HOAc peak at δ 1.69), 1.63-0.55 (theo. 105H,obs'd. 96H).

Example 66

Synthesis ofmPEG12K-b-Poly-(d-Glu(NHOH)₅-co-Glu(NHOH)₅)-b-Poly-(d-Tyr(OH)₂₀-co-Tyr(OH)₂₀)-Ac

Using the method described above in Example 52,mPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly-(d-Tyr(OH)₂₀-co-Tyr(OH)₂₀)-Acwas converted to the title compound using lithium hydroxide monohydrate(2.0 equiv./Bn ester moiety). Reaction time was 5.5 hours. Workupfollowed by precipitation with IPA, TBME afforded the title compound(Yield=93.2%) as a fine, colorless solid with a slight odor of aceticacid. ¹H NMR (d₆-DMSO) δ 9.55 (theo. 40H, obs'd. 26H), 8.45-7.90 (theo.50H, obs'd. 34H), 7.37-6.51 (theo. 160H, obs'd. 166H), 4.55-4.10 (theo.50H, obs'd. 50H), 3.80-3.20 (theo. 1087H, obs'd. 1269H, containsobscured water peak), 3.00-2.20 (theo. 80H, obs'd. 108H), 2.18-1.60(theo. 40H, obs'd. 39H, contains overlapping HOAc peak at δ 1.69).

Example 67

Synthesis ofmPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

By using the method of Example 43, reaction ofmPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OBn)₂₅-co-d-Phe₁₅)-Acwith PMB in TFA for 3.5 hours at room temperature gave a crude product,which was dissolved in dichloromethane (2 volumes) and then precipitatedfrom TBME (5 volumes). Filtration and drying in vacuo afforded the titleproduct (Yield=93.1%) as a fine, colorless, odorless solid. ¹H NMR(d₆-DMSO): identical to Example 37.

Example 68

Synthesis ofmPEG12K-b-Poly-[d-Glu(NHOH)₅-co-Glu(NHOH)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

mPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac(38.94 g, 1.90 mmol) was dissolved in 390 mL of THF and treated withhydroxylamine solution (25.2 mL, 380.0 mmol) and 4M potassium hydroxidesolution (9.5 mL, 38.0 mmol, 2.0 equiv./Bn ester moiety). The resultantslightly hazy pale yellow solution was stirred at room temperature for5.5 hours under N₂ and then diluted with acetone (220.7 g, 3.8 mol, 280mL). Acetic acid (22.82 g, 380.0 mmol, 21.7 mL) was added, the solutionwas briefly heated to reflux, and then was stirred at room temperaturefor 18 hours. The solution was diluted with 280 mL of acetone and theproduct was precipitated by addition of TBME (5 L) and diethyl ether (1L) using vigorous mechanical stirring. After cooling to −25° C. andstirring an additional 30 minutes, filtration and drying in vacuoafforded the title compound (35.98 g, Yield=87.3%) as a fine, colorlesssolid with a slight odor of acetic acid. ¹H NMR (d₆-DMSO): identical toExample 38.

Example 69

Synthesis ofmPEG11K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly-(d-Phe₁₅-co-Tyr(OBn)₂₅)-Ac

Utilizing the dichloromethane, DMAC co-solvent method detailed inExample 58 with m-PEG11k-NH₂ (1.10 kg, 100.0 mmol) and the appropriateNCA building blocks afforded a crude polymer solution in DMAC that wasprecipitated with 8 volumes of isopropanol. After filtration, the crudeproduct was slurried in 5 volumes of isopropanol for two hours. Theresultant solid was filtered, washed with fresh IPA/Et₂O, Et₂O and thenvacuum oven dried overnight to afford 2130 g (97.8% yield) of product asa nearly colorless, odorless solid. ¹H-NMR (d₆-DMSO) δ 8.45-7.85 (theo.50H, obs'd. 50H), 7.45-6.60 (theo. 350H, obs'd. 350H), 5.10-4.84 (theo.70H, obs'd. 68H), 4.65-4.20 (theo. 50H, obs'd. 48H), 3.72-3.25 (theo.1000H, obs'd. 1120H), 3.05-2.55 (theo. 50H, obs'd. 49H), 2.44-1.60(theo. 70H, obs'd. 68H).

Example 70

Synthesis ofmPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

By using the method of Example 37, reaction ofmPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OBn)₂₅-co-d-Phe₁₅)-Acwith PMB in TFA for three hours at room temperature and precipitationfrom a mixture of dichloromethane, TBME: 1, 5 afforded the title product(Yield=92.7%) as a fine, colorless, odorless polymer. ¹H NMR (d₆-DMSO)identical to Example 37.

Example 71

Synthesis ofmPEG12K-b-Poly-[d-Glu(NHOH)₅-co-Glu(NHOH)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

Using the method described in Example 52,mPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Acwas converted to the title compound using hydroxylamine solution (5equiv./ester moiety) and 4M potassium hydroxide solution (2.0 equiv./Bnester moiety). Reaction time was 5.25 hours. Acetone/acetic acid workup,precipitation with IPA, TBME: 1, 2 and further trituration of the filtercake with IPA, TBME: 1, 2 and vacuum drying afforded the title compound(Yield=89.9%) as a cream-colored solid with a slight odor of aceticacid. ¹H NMR (d₆-DMSO): identical to Example 38.

Example 72

Synthesis ofmPEG12K-b-Poly-[d-Glu(NHOH)₅-co-Glu(NHOH)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

Using the method described in Example 71,mPEG12K-b-Poly-[d-Glu(OBn)₅-co-Glu(OBn)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Acwas converted to the title compound using hydroxylamine solution (10equiv./ester moiety) and 4M potassium hydroxide solution (2.0 equiv./Bnester moiety). Reaction time was 5.5 hours. Acetone/acetic acid workup,precipitation with IPA, TBME: 1, 4 and vacuum drying afforded the titlecompound (Yield=82.9%) as a fine, colorless solid with a slight odor ofacetic acid. ¹H NMR (d₆-DMSO): identical to Example 38.

Example 73

Synthesis of mPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(Tyr(OBn)₁₀-co-d-Phe₂₀-co-Asp(oTbu)₁₀-Ac

mPEG12K-NH2 prepared in the same manner as Example 3, was weighed (30 g,2.5 mmol) into a clean 500 mL round bottom flask and dissolvedcompletely in toluene and dried by azeotropic distillation. Toluene wascollected into a second 500 mL round bottom flask chilled with nitrogen,via a simple glass bridge. Resultant solid was allowed to dry completelyfor three hours. To the dry solid freshly distilled N-methylpyrrolidinewas added via cannula and vacuum transfer. This mixture was allowed todissolve completely before the addition NCA. The NCA as prepared fromexample 8 and example 9 accordingly, was weighed out into a clean twoneck round bottom flask Glu(oBn) NCA (2.87 g) d-Glu(oBn) NCA (2.87 g)and evacuated for one hour before this solid was dissolved completely inNMP, and then cannulated into the flask containing the PEG. Thispolymerization was stirred at room temperature and monitored by GPC(DMF, 0.1% LiBr) to ensure completion (about 16 hrs). Upon completion ofpolymerization of this first block of NCA, the second second addition ofNCA was done in the same manner as the first, and consisted of d-Phe(9.5 g) from example 7, Tyr(oBn) (7.4 g) from example 6, and Asp(otBu)(5.38 g) from example 5. This was allowed to polymerize at roomtemperature for two hours and then heated to 35° C. until completion(about 24 hrs). Once confirmed by GPC, N-Methyl-Morpholine (2.5 g, 2.7mL, 25 mmol), DMAP (0.3 g, 2.5 mmol), and Acetic Anhydride (2.5 g, 2.36mL, 25 mmol), was added to the reaction solution was stirred overnight.This reaction mixture was poured into a two liter beaker with a magneticstir-bar, and diethyl ether was slowly added until a white precipitatewas observed. This solid was filtered and washed on a medium porositysintered glass frit. This solid was dried in vacuo, characterized with¹H NMR and GPC. (yield=74.8%, 40 grams). ¹H NMR (d₆-DMSO) δ 8.42-7.70,7.30, 6.95, 5.10-4.9, 4.65-4.20, 3.77-3.25, 3.05-2.45, 2.44-1.60,1.38-1.22.

Example 74

Synthesis of mPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(Tyr₁₀-co-d-Phe₂₀-co-Asp₁₀-Ac

The Protected Triblock co-polymer (Example 73) was weighed (30 g, 1.4mmol) into a clean 500 mL beaker and dissolved in triflouroacetic acid.Pentamentylbenzene (4 g, 26.98 mmol) was added and stirred with amagnetic stir-bar. The reaction mixture was stirred for two hours andmonitored by NMR for complete removal of benzylic protecting groups ontyrosine and t-Butyl group on aspartate. After completion of thisdeprotection the solution was precipitated in cold diethyl ether. Thissolid was then filtered on a medium sintered glass frit and re-dissolvedin methylene chloride and agin precipitated in cold ether and filtered.This solid (24.7 g, Yield=88.4%) was dried in vacuo and characterized.¹H NMR (d6-DMSO) δ 9.09, 8.50-7.75, 7.40-6.45, 5.03, 4.70-4.20,3.91-3.05, 3.03-2.10, 2.09-1.50.

Example 75

Synthesis of mPEG12K-b-Poly-(d-Glu(oBn)₅-co-Glu(oBn₅)-b-Poly(Tyr(OBn)₁₀-co-d-Leu₂₀-co-Asp(oTbu)₁₀-Ac

Using the general protocol from Example 73 and substituting appropriateNCA starting materials resulted in the crude polymer, this wasprecipitated with diethyl ether about 10 volumes. After filtration anddrying the title compound (Yield=80.2%) was collected as a colorlesssolid. ¹H NMR (d6-DMSO) δ 8.50-7.75, 7.40-6.6, 5.03, 4.70-4.20,3.69-3.09, 3.03-2.10, 2.09-1.50, 1.43-1.25, 0.85-0.62.

Example 76

Synthesis of mPEG12K-b-Poly-(d-Glu(oBn)₅-co-Glu(oBn)₅-b-Poly(Tyr(OH)₁₀-co-d-Leu₂₀-co-Asp₁₀)-Ac

Thirty four grams of the protected triblock polymer (Example 75) wasweighed into a clean 500 mL beaker and dissolved in triflouroacetic acid(500 mL). To this solution (4 g, 27 mmol) pentamentyl-benzene was addedand stirred with a magnetic stir-bar. At thirty mins post addition ofpentamethyl-benzene a precipitate was observed in solution. The reactionmixture was stirred for 2.5 hours and monitored by NMR for completeremoval of benzylic protecting groups on tyrosine and t-Butyl group onaspartate. After completion of this deprotection the solution wasrotovapped to a thick paste, redissolved in methylene chloride and thenprecipitated in cold diethyl ether. This solid was then filtered on amedium sintered glass frit and re-dissolved in methylene chloride andagin precipitated in cold ether and filtered. This solid was dried undervacuum and characterized. ¹H NMR (d6-DMSO) δ 9.09, 8.50-7.75, 7.45-6.55,5.03, 4.65-4.00, 3.69-3.09, 3.03-2.10, 2.09-1.50, 0.85-0.55.

Example 77

Synthesis of mPEG12K-b-(d-Glu(NHOH)₅-co-Glu(NHOH)-b-Poly(Tyr(OH)₁₀-co-d-Leu₂₀-co-Asp₁₀)-Ac

Triblock ester (Example 76) was weighed (20 g, 0.96 mmol) into a clean500 mL round bottom flask and 200 mL of tetrahydrofuran was added anddissolved completely. To this solution thirty equivalents ofhydroxylamine (1.9 mL, 28 mmol) and 0.5 g of TBD catalyst was stirredunder nitrogen at 50° C. overnight. Completion was verified by ¹H NMR.This solution was mixed with 100 mL methanol and precipitated withdiethyl ether (about 7 volumes). This white solid was collected byfiltration and washed with fresh diethyl ether. The collected solid wasthen dissolved in acetone and and a catalytic amount of acetic acid wasallowed to stir overnight. The solution was poured into a clean twoliter beaker and diethyl ether was slowly added to the solution withstirring. ¹H NMR (d6-DMSO) δ 9.4-8.6, 8.51-7.77, 7.44-7.57, 6.96, 6.56,4.52-4.00, 3.75-3.29, 3.03-2.45, 2.08-1.21, 0.95-0.57.

Example 78

Synthesis of mPEG12K-b-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(Tyr(OBn)₃₀-co-d-Phe₁₀)-Ac

The first block of the copolymer was prepared using the same scale andprocedure as example 73. Upon completion of this first block of NCA asecond addition of NCA of d-Phe NCA (4.78 g, 25 mmol) prepared in thesame manner as example 7, and of Tyr(oBn) NCA (22.29 g, 75 mmol) fromthe procedure in example 6. This solution was allowed to polymerize atroom temperature for two hours and then heated to 35° C. untilcompletion (about 48 hrs). Once confirmed by GPC, N-Methyl-Morpholine(2.5 g, 2.7 mL, 25 mmol), DMAP (0.3 g, 2.5 mmol), and Acetic Anhydride(2.5 g, 2.36 mL, 25 mmol), was added to the reaction solution wasstirred overnight. This capped polymer was worked up in the same manneras in Example 73. (yield=79.6%) about 40 grams. ¹H NMR (d6-DMSO) δ8.46-7.72, 7.44-6.57, 5.10-4.80, 4.62-4.13, 3.74-3.23, 3.03-2.77,2.62-2.21, 2.02-1.56 (solvent impurities).

Example 79

Synthesis of mPEG12K-b-Poly-(d-Glu(oBn)₅-co-Glu(oBn)₅-b-Poly(Tyr(OH)₃₀-co-d-Phe₁₀)-Ac

The protected triblock co-polymer (from Example 78) was weighed (34 g,1.46 mmol) into a clean 500 mL beaker and dissolved in triflouroaceticacid (500 mL). To this solution (4 g, 27 mmol) pentamentyl-benzene wasadded and stirred with a magnetic stir-bar. At thirty minutes postaddition of pentamethyl-benzene a precipitate was observed in solution.The reaction mixture was stirred for 2.5 hours and monitored by NMR forcomplete removal of benzylic protecting groups on tyrosine. Aftercompletion of this deprotection (3 hrs) the solution was rotovapped to athick paste, redissolved in methylene chloride and then precipitated incold diethyl ether. This solid was then filtered on a medium sinteredglass frit and re-dissolved in methylene chloride and agin precipitatedin cold ether and filtered. This solid was dried in vacuo andcharacterized. ¹H NMR (d6-DMSO) δ 9.10, 8.38-7.77, 7.39-6.73, 6.59,5.03, 4.64-3.79, 3.71-3.30, 2.98-2.56, 2.02-1.62.

Example 80

Synthesis of mPEG12K-b-Poly-(d-Glu(NHOH)₅-co-Glu(NHOH)₅)-b-Poly(Tyr(OH)₃₀-co-d-Phe₁₀)-Ac

20 g of Triblock ester (From Example 79) was weighed into a clean 500 mLround bottom flask and 200 mL of tetrahydrofuran was added and dissolvedcompletely. To this solution ten equivalents of hydroxylamine, and1,5,7-triazabicyclo [4.4.0]dec-5-ene (TBD, 0.5 g, 3.5 mmol) was stirredunder nitrogen at room temperature. Completion was verified by ¹H NMR(48 Hrs). This solution was mixed with 100 mL methanol and this solutionwas poured into a clean two liter beaker. Methyltertbutyl ether (about 5volumes) was slowly added to the solution with stirring. The resultantwhite solid was then collected on a medium frit and dried in vacuo.(17.34 g, Yield=90%). ¹H NMR (d₆-DMSO) δ 9.10-8.65, 8.39-7.78,7.28-6.75, 6.80, 6.59, 4.59-4.31, 3.75-3.13, 3.00-2.57, 2.16-1.57.

Example 81

Synthesis of mPEG12K-b-Poly-(d-Glu(OBn)_(1.5)-co-Glu(OBn)_(1.5))-b-Poly(Tyr(OBn)₂₅-co-d-Phe₁₅)-Ac

mPEG12KNH₂ prepared in the same manner as example 3 (25 g, 2.08 mm) wasweighed into a clean, oven dried, 1000 mL, two neck, round bottom flaskand dissolved in toluene (300 mL) with heating and dried by azeotropicdistillation. After distillation to dryness, the polymer was left undervacuum for three hours. The flask was subsequently backfilled with N₂,re-evacuated under reduced pressure, and dry N-methylpyrrolidone (NMP)(250 mL) was introduced by cannula. The mixture was briefly heated to40° C. to expedite dissolution and then cooled to 25° C. Glu(OBn) NCA(0.82 g, 3.1 mmol) made in the same manner as example 8, and d-Glu(OBn)NCA (0.82 g, 3.1 mmol) from example 9, were added to the flask directly,and the reaction mixture was allowed to stir for 18 hours at roomtemperature under nitrogen gas. Then, d-Phe NCA (5.97 g, 31.25 mmol)from example 7, and Tyr(OBn) NCA (15.49 g, 52.08 mmol) prepared fromexample 6, and were then added to the solution and stirred for 2 hoursthen heated to 35° C. for 48 hours at which point the reaction wascomplete (GPC, DMF/0.1% LiBr). The solution was cooled to roomtemperature and acetic anhydride (2.04 g, 20 mmol, 1.88 mL),N-methylmorpholine (NMM) (2.23 g, 22 mmol, 2.47 mL) anddimethylaminopyridine (DMAP) (0.24 g, 2.0 mmole) were added. Stirringwas continued for 1 day at room temperature. The polymer wasprecipitated into diethyl ether:heptane 10:1 (2.5 L) and isolated byfiltration, washed with fresh 100 mL portions of diethyl ether, anddried in vacuo to give the block copolymer as a fine, off white powder(39.81 g, Yield=90.3%). ¹H NMR (d₆-DMSO) δ 9.26-9.04, 8.36-7.75,7.41-7.25, 6.97, 6.60, 5.04, 4.59-4.13, 3.81-3.13, 2.96-2.76, 2.75-2.57,2.43-2.12, 2.00-1.45.

Example 82

Synthesis of mPEG12K-b-Poly-(d-Glu(OBn)_(1.5)-co-Glu(OBn)_(1.5))-b-Poly(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

The polymer from Example 81 was deprotected using the general methodfrom example 74 only adjusting stoichometry. Once complete (3 hrs) thesolution was rotovapped to a thick paste and then redissolved indicholomethane and precipitated in cold Diethyl ether, collected byfiltration and dried in vacuo. This reaction yielded 22 g of drymaterial (Yield=76.92%). ¹H NMR (d6-DMSO) δ 9.09, 8.50-7.75, 7.35-6.45,5.04, 4.70-4.20, 3.91-3.05, 3.03-2.10, 2.09-1.50.

Example 83

Synthesis ofmPEG12K-b-Poly-(d-Glu(NHOH)_(1.5)-co-Glu(NHOH)_(1.5))-b-Poly(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

The polymer from Example 82 (13.2 g, 0.705 mmol) was dissolvedcompletely in 160 mL of THF with heating, this solution was allowed tocool to room temp before 1,5,7-triazabicyclo [4.4.0]dec-5-ene (TBD, 0.3g, 2.2 mmol) was added followed by Hydroxylamine (50% water solution, 25mL, 378 mmol) this solution was stirred at room temperature for 24hours. Methanol (80 mL) was added and then precipitated withmethyltertbutyl ether, collected by filtration, and dissolved inacetone. Acetic acid was added to this acetone solution and stirred for5 hours. The solution was evaporated until nearly dry, redissolved inmethylene chloride and precipitated in MTBE, collected by filtration anddried in vacuo (12.1 g, Yield=92.8%). ¹H NMR (d₆-DMSO) δ 9.11,8.34-7.75, 7.37-7.05, 6.92, 6.58 4.60-4.32, 3.81-3.12, 2.99-2.57,2.49-2.32, 2.10-1.73.

Example 84

Synthesis ofmPEG12K-b-Poly-(d-Glu(OBn)_(2.5)-co-Glu(OBn)_(2.5))-b-Poly(Tyr(OBn)₂₅-co-d-Phe₁₅)-Ac

mPEG12KNH₂ prepared from the same method as Example 3, (25 g, 2.08 mm)was weighed into a clean, oven dried, 1000 mL, two neck, round bottomflask and dissolved in toluene (300 mL) with heating and dried byazeotropic distillation. After distillation to dryness, the polymer wasleft under vacuum for three hours. The flask was subsequently backfilledwith N₂, re-evacuated under reduced pressure, and dryN-methylpyrrolidone (NMP) (250 mL) was introduced by cannula. Themixture was briefly heated to 40° C. to expedite dissolution and thencooled to 25° C. Glu(OBn) NCA (1.37 g, 5.2 mmol) and d-Glu(OBn) NCA(1.37 g, 5.2 mmol) were added to the flask, and the reaction mixture wasallowed to stir for 18 hours at room temperature under nitrogen gas.After completion of the first block of NCA, d-Phe NCA (5.97 g, 31.25mmol) prepared in the same manner as Example 79, and Tyr (OBn) NCA(15.49 g, 52.08 mmol) from example 6, were added and the solution wasallowed to stir at room temperature for two hours at 35° C. for 48 hoursat which point the reaction was complete (GPC, DMF/0.1% LiBr). Thesolution was cooled to room temperature and acetic anhydride (2.04 g, 20mmol, 1.88 mL), N-methylmorpholine (NMM) (2.23 g, 22 mmol, 2.47 mL) anddimethylaminopyridine (DMAP) (0.24 g, 2.0 mmole) were added. Stirringwas continued for 1 day at room temperature. The polymer wasprecipitated into diethyl ether:heptane 10:1 (2.5 L) and isolated byfiltration, washed with fresh 100 mL portions of diethyl ether, anddried in vacuo to give the block copolymer as a fine, off white powder(36 g, Yield=80%). ¹H NMR (d₆-DMSO) δ 9.10 8.37-7.83, 7.39-7.21, 6.95,6.56, 5.02, 4.61-4.34, 4.32-4.20, 3.71-3.25, 2.94-2.59, 2.40-2.10,1.96-1.45.

Example 85

Synthesis of mPEG12K-b-Poly-(d-Glu(OBn)_(2.5)-co-Glu(OBn)_(2.5))-b-Poly(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

Using the general method from Example 74 and adjusting stoichiometry,the polymer from Example 84 was deprotected (32 g, 1.65 mmol). Oncecomplete (3 Hrs.) the solution was rotovapped to a thick paste and thenredissolved in DCM and precipitated in cold Diethyl ether, collected byfiltration and dried in vacuo. This reaction yielded 27 g of drymaterial (94.2%). ¹H NMR (d6-DMSO) δ 9.09, 8.50-7.75, 7.35-6.45, 5.04,4.70-4.20, 3.91-3.05, 3.03-2.10, 2.09-1.50.

Example 86

Synthesis ofmPEG12K-b-Poly-(d-Glu(NHOH)_(2.5)-co-Glu(NHOH)_(2.5))-b-Poly(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

Polymer from Example 85 (20 g, 1 mmol) dissolved completely in 160 mL ofTHF with heating, this solution was allowed to cool to room temp before1,5,7-triazabicyclo [4.4.0]dec-5-ene (TBD, 0.5 g, 3.6 mmol) was added,followed by Hydroxylamine (50% water solution, 30 mL, 545 mmol) thissolution was stirred at room temperature for 24 hours. Methanol (80 mL)was added and then precipitated with methyltertbutyl ether, collected byfiltration, and dissolved in acetone. Acetic acid was added to thisacetone solution and stirred for 5 hours, then this solution wasrotovapped until nearly dry, redissolved in methylenechloride andprecipitated in MTBE, collected by filtration and dried in vacuo (18 g,Yield=91.7%). ¹H NMR (d₆-DMSO) δ 9.11, 8.34-7.75, 7.15, 6.80, 4.60-4.32,3.81-3.12, 2.99-2.32, 1.93-1.83).

Example 87

Synthesis of mPEG12K-b-Poly-(d-Glu(OBn)_(3.5)-co-Glu(OBn)_(3.5))-b-Poly(Tyr(OBn)₂₅-co-d-Phe₁₅)-Ac

mPEG12KNH₂ prepared in the same manner as example 3 (25 g, 2.08 mm) wasweighed into a clean, oven dried, 1000 mL, two neck, round bottom flaskand dissolved in toluene (300 mL). This polymer was prepared in the samemanner as example 1. Glu(OBn) NCA (1.92 g, 7.3 mmol) from Example 8 andd-Glu(OBn) NCA (1.92 g, 7.3 mmol) from Example 9, were added to theflask, and the reaction mixture was allowed to stir for 18 hours atambient room temperature under nitrogen gas. Then, d-Phe NCA (5.97 g,31.25 mmol) from Example 7 and Tyr (OBn) NCA (15.49 g, 52.08 mmol)prepared in the same way as example 6, were added and the solution wasallowed to stir at room temp for 2 hours and then heated to 35° C. for48 hours at which point the reaction was complete (GPC, DMF/0.1% LiBr).The solution was cooled to room temperature and acetic anhydride (2.04g, 20 mmol, 1.88 mL), N-methylmorpholine (NMM) (2.23 g, 22 mmol, 2.47mL) and dimethylaminopyridine (DMAP) (0.24 g, 2.0 mmole) were added.Stirring was continued for 1 day at room temperature. The polymer wasprecipitated into diethyl ether:heptane 10:1 (2.5 L) and isolated byfiltration, washed with fresh 100 mL portions of diethyl ether, anddried in vacuo to give the block copolymer as a fine, nearly colorlesspowder (37.0 g, Yield=80.6%). ¹H NMR (d₆-DMSO) δ 9.08 8.42-7.70, 7.29,6.96, 6.58, 5.10-4.85, 4.65-4.20, 3.71-3.25, 2.94-2.59, 2.40-2.10,1.97-1.50.

Example 88

Synthesis of mPEG12K-b-Poly-(d-Glu(OBn)_(3.5)-co-Glu(OBn)_(3.5))-b-Poly(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

The polymer from Example 87 was deprotected using the general methodfrom Example 74 only adjusting stoichometry (32 g, 1.61 mmol). Oncecomplete (3 Hrs.) the solution was rotovapped to a thick paste and thenredissolved in DCM and precipitated in cold Diethyl ether, collected byfiltration and dried in vacuo. This reaction yielded 23 g of drymaterial (80%). ¹H NMR (d6-DMSO) δ 9.09, 8.50-7.75, 7.35-6.45, 5.04,4.70-4.20, 3.91-3.05, 3.03-2.10, 2.09-1.50.

Example 89

Synthesis ofmPEG12K-b-Poly-(d-Glu(NHOH)_(3.5)-co-Glu(NHOH)_(3.5))-b-Poly(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

The polymer (20 g, 1 mmol) from Example 88 was dissolved completely in160 mL of THF with heating, this solution was allowed to cool to roomtemp before 1,5,7-triazabicyclo [4.4.0]dec-5-ene (TBD, 0.5 g, 3.6 mmol)was added, followed by Hydroxylamine (50% water solution, 30 mL, 545mmol) this solution was stirred at room temperature for 24 hours.Methanol (80 mL) was added and then precipitated with methyltertbutylether, collected by filtration, and dissolved in acetone. Acetic acidwas added to this acetone solution and stirred overnight. The solutionwas rotovapped until nearly dry, redissolved in methylene chloride andprecipitated in MTBE, collected by filtration and dried in vacuo (18.1g, Yield=92.9%). ¹H NMR (d₆-DMSO) δ 9.11, 8.34-7.75, 7.15, 6.80,4.60-4.32, 3.81-3.12, 2.99-2.32, 1.93-1.83.

Example 90

Synthesis of mPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(Tyr(OBn)₂₅-co-d-Phe₁₅)-Ac

mPEG12KNH₂ prepared by the same method as Example 3, was weighed (25 g,2.08 mm) into a clean, oven dried, 1000 mL, two neck, round bottom flaskand dissolved in toluene (300 mL) This polymer was prepared in the samemanner as Example 73. Then Glu(OBn) NCA (2.74 g, 10.4 mmol) prepared inthe same manner as Example 8, and d-Glu(OBn) NCA (2.74 g, 10.4 mmol)prepared in the same manner as Example 9, were added to the flask, andthe reaction mixture was allowed to stir for 18 hours at ambient roomtemperature under nitrogen gas. Then, d-Phe NCA (5.97 g, 31.25 mmol)from Example 7 and Tyr (OBn) NCA (15.49 g, 52.08 mmol) prepared from themethod in Example 6, were added and the solution was allowed to stir atroom temp for 2 hours and then heated to 35° C. for 48 hours at whichpoint the reaction was complete (GPC, DMF/0.1% LiBr). The solution wascooled to room temperature and acetic anhydride (2.04 g, 20 mmol, 1.88mL), N-methylmorpholine (NMM) (2.23 g, 22 mmol, 2.47 mL) anddimethylaminopyridine (DMAP) (0.24 g, 2.0 mmole) were added. Stirringwas continued for 1 day at room temperature. The polymer wasprecipitated into diethyl ether:heptane 10:1 (2.5 L) and isolated byfiltration, washed with fresh 100 mL portions of diethyl ether, anddried in vacuo to give the block copolymer as a fine, nearly colorlesspowder (38.48 g, Yield=81.4%). ¹H NMR (d₆-DMSO) δ 9.08 8.42-7.70, 7.29,6.97, 5.11-4.84, 4.65-4.20, 3.72-3.25, 3.05-2.45, 2.44-1.59.

Example 91

Synthesis of mPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

Using the general method from example 74 only adjusting stoichometrythis polymer was deprotected (32 g, 1.56 mmol). Once complete (3 Hrs.)the solution was rotovapped to a thick paste and then redissolved in DCMand precipitated in cold Diethyl ether, collected by filtration anddried in vacuo. This reaction yielded 27 g of dry material (93.6%). ¹HNMR (d6-DMSO) δ 9.09, 8.50-7.75, 7.35-6.45, 5.04, 4.70-4.20, 3.91-3.05,3.03-2.10, 2.09-1.50.

Example 92

Synthesis of mPEG12K-b-Poly-(d-Glu(NHOH)₅-co-Glu(NHOH)₅)-b-Poly(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

The polymer (18 g, 0.88 mmol) from Example 91 was dissolved completelyin 160 mL of THF with heating, this solution was allowed to cool to roomtemp before 1,5,7-triazabicyclo [4.4.0]dec-5-ene (TBD, 0.5 g, 3.6 mmol)was added, followed by Hydroxylamine (50% water solution, 30 mL, 545mmol) this solution was stirred at room temperature for 24 hours.Methanol (80 mL) was added and then precipitated with methyltertbutylether, collected by filtration, and dissolved in acetone. Acetic acidwas added to this acetone solution and stirred for 5 hours and thenworked up. The solution was rotovapped until nearly dry, redissolved inmethylene chloride and precipitated in MTBE, collected by filtration anddried in vacuo (16.7 g, Yield=96.3%).

Example 93

Synthesis of mPEG12K-b-Poly-(d-Glu(OBn)_(7.5)-co-Glu(OBn)_(7.5))-b-Poly(Tyr(OBn)₂₅-co-d-Phe₁₅)-Ac

mPEG12KNH₂ (25 g, 2.08 mm) prepared in the same manner as Example 3, wasweighed into a clean, oven dried, 1000 mL, two neck, round bottom flaskand dissolved in toluene (300 mL) This polymer was prepared in the samemanner as example 1. Glu(OBn) NCA (2.74 g, 10.4 mmol) prepared in thesame way as example 8 and d-Glu(OBn) NCA (2.74 g, 10.4 mmol) fromExample 9, were added to the flask, and the reaction mixture was allowedto stir for 18 hours at room temperature under nitrogen gas. Then, d-PheNCA (5.97 g, 31.25 mmol) from Example 7 and Tyr (OBn) NCA (15.49 g,52.08 mmol) from Example 6, were added and the solution was allowed tostir at room temp for 2 hours and then heated to 35° C. for 48 hours atwhich point the reaction was complete (GPC, DMF/0.1% LiBr). The solutionwas cooled to room temperature and acetic anhydride (2.04 g, 20 mmol,1.88 mL), N-methylmorpholine (NMM) (2.23 g, 22 mmol, 2.47 mL) anddimethylaminopyridine (DMAP) (0.24 g, 2.0 mmole) were added. Stirringwas continued overnight at room temperature. The polymer wasprecipitated into diethyl ether:heptane 10:1 (2.5 L) and isolated byfiltration, washed with fresh 100 mL portions of diethyl ether, anddried in vacuo to give the block copolymer as a nearly colorless powder(37.0 g, Yield=74.66%). ¹H NMR (d₆-DMSO) δ 9.10 8.42-7.71, 7.27, 6.97,5.11-4.85, 4.65-4.20, 3.72-3.25, 3.05-2.45, 2.45-1.60.

Example 94

Synthesis of mPEG12K-b-Poly-(d-Glu(OBn)_(7.5)-co-Glu(OBn)_(7.5))-b-Poly(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

Using the general method from Example 74 only adjusting stoichometrythis polymer was deprotected (32 g, 1.48 mmol). Once complete (3 Hrs.)the solution was rotovapped to a thick paste and then redissolved in DCMand precipitated in cold Diethyl ether, collected by filtration anddried in vacuo. This reaction yielded 24 g of dry material (82.8%). ¹HNMR (d6-DMSO) δ 9.09, 8.50-7.75, 7.35-6.45, 5.04, 4.70-4.20, 3.91-3.05,3.03-2.10, 2.09-1.50.

Example 95

Synthesis ofmPEG12K-b-Poly-(d-Glu(NHOH)_(7.5)-co-Glu(NHOH)_(7.5))-b-Poly(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

The polymer (19 g, 0.88 mmol) prepared in Example 94 was dissolvedcompletely in 160 mL of THF with heating, this solution was allowed tocool to room temp before 1,5,7-triazabicyclo [4.4.0]dec-5-ene (TBD, 0.5g, 3.6 mmol) was added, followed by Hydroxylamine (50% water solution,30 mL, 545 mmol) this solution was stirred at room temperature for 24hours. Methanol (80 mL) was added and then precipitated withmethyltertbutyl ether, collected by filtration, and dissolved inacetone. Acetic acid was added to this acetone solution and stirred for5 hours. The solution was rotovapped until nearly dry, redissolved inmethylene chloride and precipitated in MTBE, collected by filtration anddried in vacuo (16.4 g, Yield=91.1%). ¹H NMR (d₆-DMSO) δ 9.11,8.34-7.75, 7.15, 6.80, 4.60-4.32, 3.81-3.12, 2.99-2.32, 1.93-1.83.

Example 96

Synthesis of mPEG12K-b-Poly-(d-Glu(OBn)₁₀-co-Glu(OBn)₁₀)-b-Poly(Tyr(OBn)₂₅-co-d-Phe₁₅)-Ac

mPEG12KNH₂ (25 g, 2.08 mm) prepared in the same was as Example 3, wasweighed into a clean, oven dried, 1 L, two neck, round bottom flask anddissolved in toluene (300 mL) This polymer was prepared in the samemanner as example 1. Glu(OBn) NCA (5.48 g, 20.8 mmol) prepared in thesame manner as Example 8, and d-Glu(OBn) NCA (5.48 g, 20.8 mmol)prepared by the method in Example 9, were added to the flask directly,and the reaction mixture was allowed to stir for 16 hours at ambientroom temperature under nitrogen gas. Then, d-Phe NCA (5.97 g, 31.25mmol) from Example 7 and Tyr (OBn) NCA (15.49 g, 52.08 mmol) fromExample 6, were added and the solution and allowed to stir at room tempfor 2 hours and then heated to 35° C. for 48 hours at which point thereaction was complete (GPC, DMF/0.1% LiBr). The solution was cooled toroom temperature and acetic anhydride (2.04 g, 20 mmol, 1.88 mL),N-methylmorpholine (NMM) (2.23 g, 22 mmol, 2.47 mL) anddimethylaminopyridine (DMAP) (0.24 g, 2.0 mmole) were added. Stirringwas continued for 1 day at room temperature. The polymer wasprecipitated into diethyl ether:heptane 10:1 (2.5 L) and isolated byfiltration, washed with fresh 100 mL portions of diethyl ether, anddried in vacuo to give the block copolymer as a fine, nearly colorlesspowder (38.9 g, Yield=75.23%). ¹H NMR (d₆-DMSO) δ 9.08, 8.40-7.65,7.35-7.25, 6.99, 6.76, 5.10-4.85, 4.65-4.20, 3.72-3.25, 3.06-2.45,2.34-1.59.

Example 97

Synthesis of mPEG12K-b-Poly-(d-Glu(OBn)₁₀-co-Glu(OBn)₁₀)-b-Poly(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

Using the general method from Example 74 only adjusting stoichometrythis polymer was deprotected (32 g, 1.41 mmol). Once complete (3 Hrs.)the solution was rotovapped to a thick paste and then redissolved in DCMand precipitated in cold Diethyl ether, collected by filtration anddried in vacuo. This reaction yielded 27 g of dry material (92.8%). ¹HNMR (d6-DMSO) δ 9.09, 8.50-7.75, 7.35-6.45, 5.04, 4.70-4.20, 3.91-3.05,3.03-2.10, 2.09-1.50.

Example 98

Synthesis of mPEG12K-b-Poly-(d-Glu(NHOH)₁₀-co-Glu(NHOH)₁₀)-b-Poly(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

The polymer (20 g, 0.88 mmol) from Example 98 was dissolved completelyin 160 mL of THF with heating, this solution was allowed to cool to roomtemp before 1,5,7-triazabicyclo [4.4.0]dec-5-ene (TBD, 0.5 g, 3.6 mmol)was added, followed by Hydroxylamine (50% water solution, 30 mL, 545mmol) this solution was stirred at room temperature for 24 hours.Methanol (80 mL) was added and then precipitated with methyltertbutylether, collected by filtration, and dissolved in acetone. Acetic acidwas added to this acetone solution and stirred for 5 hours and thenworked up. The solution was rotovapped until nearly dry, redissolved inmethylenechloride and precipitated in MTBE, collected by filtration anddried in vacuo (17.2 g, Yield=92.1%). ¹H NMR (d₆-DMSO) δ 9.11,8.33-7.69, 7.15, 6.98, 6.79, 5.06-4.85, 4.60-4.32, 3.81-3.19, 2.99-2.32,2.03-1.59.

Example 99

Synthesis of mPEG12K-b-Poly-(d-Glu(OBn)_(3.5)-co-Glu(OBn)_(3.5))-b-Poly(Tyr(OBn)₂₅-co-d-Phe₁₅)-Ac

mPEG12KNH₂ (45.34 g, 3.78 mmol) prepared by the same method detailed inExample 3, was weighed into a clean, oven dried, 1000 mL, two neck,round bottom flask and dissolved in toluene (300 mL) This polymer wasprepared in the same manner as Example 73. Glu(OBn) NCA (3.5 g, 13.29mmol) from the method detailed in Example 8, and d-Glu(OBn) NCA (3.5 g,13.29 mmol) from the method detailed in Example 9, were added to theflask, and the reaction mixture was allowed to stir for 16 hours atambient room temperature under nitrogen gas. Then, d-Phe NCA (10.89 g,56.9 mmol) from the method detailed in Example 7, and Tyr (OBn) NCA(28.24 g, 94.98 mmol) from the method detailed in Example 6, were addedand the solution was allowed to stir at 35° C. for 48 hours at whichpoint the reaction was complete (GPC, DMF/0.1% LiBr). The solution wascooled to room temperature and acetic anhydride (3.88 g, 37.8 mmol, 3.58mL), N-methylmorpholine (NMM) (3.76 g, 37.8 mmol, 4.16 mL) anddimethylaminopyridine (DMAP) (0.47 g, 3.8 mmole) were added. Stirringwas continued for 1 day at room temperature. The polymer wasprecipitated into diethyl ether:heptane 10:1 (2.5 L) and isolated byfiltration, washed with fresh 100 mL portions of diethyl ether, anddried in vacuo to give the block copolymer as a fine, nearly colorlesspowder (68.22 g, Yield=82.2%). ¹H NMR (d₆-DMSO) δ 8.43-7.84, 7.30, 6.98,6.97-6.65, 5.04, 4.98-4.80, 4.66-4.16, 3.72-3.21, 3.01-2.76, 2.74-2.56,2.41-2.26, 2.23-2.10, 2.01-1.58.

Example 100

Synthesis of mPEG12K-b-Poly-(d-Glu(OBn)_(3.5)-co-Glu(OBn)_(3.5))-b-Poly(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

Using the general method from Example 74 only adjusting stoichometrythis polymer was deprotected (60 g, 2.73 mmol). Once complete (5 Hrs.)the solution was rotovapped to a thick paste and then redissolved in DCMand precipitated in cold Diethyl ether, collected by filtration, washedseveral times with fresh 200 mL portions of cold diethyl ether and driedin vacuo. This reaction yielded 43.8 g of dry material (81.4%). ¹H NMR(d6-DMSO) δ 9.04, 8.38-7.73, 7.38-6.73, 5.04, 4.62-4.19, 3.82-3.27,3.02-2.76, 2.75-2.56, 2.42-2.26, 2.20-1.61, 1.08 (solvent, ether).

Example 101

Synthesis ofmPEG12K-b-Poly-(d-Glu(NHOH)_(3.5)-co-Glu(NHOH)_(3.5))-b-Poly(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac

The polymer (40 g, 1 mmol) from Example 100, was dissolved completely in700 mL of THF with heating, this solution was allowed to cool to roomtemp before 1,5,7-triazabicyclo [4.4.0]dec-5-ene (TBD, 1.5 g, 10.8 mmol)was added, followed by Hydroxylamine (50% water solution, 45 mL, 817.5mmol) this solution was stirred at room temperature for 24 hours.Isopropanol (200 mL) was added and then precipitated withmethyltertbutyl ether, collected by filtration, and dissolved in acetone(500 mL). Acetic acid (5 mL) was added to this acetone solution andstirred overnight. The solution was rotovapped until nearly dry,redissolved in methylenechloride and precipitated in MTBE, collected byfiltration and dried in vacuo (33.5 g, Yield=86%). ¹H NMR (d₆-DMSO) δ9.03, 8.37-7.70, 7.36-6.72, 6.68-6.42, 4.64-4.14, 3.73-3.10, 3.00-2.76,2.71-2.56, 2.42-2.27, 2.21-1.61.

Example 102

Synthesis ofmPEG11.5K-b-Poly-(d-Glu(OBn)_(3.5)-co-Glu(OBn)_(3.5))-b-Poly(Tyr(OBn)₂₅-co-d-Phe₁₅)-Ac

PEG11.5KNH₂ (15 g, 1.3 mmol) prepared with the same method as Example 3with the exception of molecular weight, was weighed into a clean, ovendried, 1000 mL, two neck, round bottom flask and dissolved in toluene(300 mL) This polymer was prepared in the same manner as Example 73.Glu(OBn) NCA (1.2 g, 4.56 mmol) prepared with the same method as Example8, and d-Glu(OBn) NCA (1.2 g, 4.56 mmol) prepared with the same methodas Example 9, were added to the flask, and the reaction mixture wasallowed to stir for 16 hours at ambient room temperature under nitrogengas. Then, d-Phe NCA (2.88 g, 19.56 mmol) prepared with the same methodas Example 7, and Tyr (OBn) NCA (8.26 g, 32.60 mmol) prepared with thesame method as Example 6, were added and the solution directly, andallowed to stir at room temp for 2 hours and then heated to 35° C. for48 hours at which point the reaction was complete (GPC, DMF/0.1% LiBr).The solution was cooled to room temperature and acetic anhydride (1.34g, 13 mmol, 1.23 mL), N-methylmorpholine (NMM) (1.3 g, 13 mmol, 1.43 mL)and dimethylaminopyridine (DMAP) (0.16 g, 1.3 mmole) were added.Stirring was continued for 16 hours at room temperature. The polymer wasprecipitated into diethyl ether:heptane 10:1 (2.5 L) and isolated byfiltration, washed with fresh 100 mL portions of diethyl ether, anddried in vacuo to give the block copolymer as a fine, nearly colorlesspowder (26 g, Yield=82.2%). ¹H NMR (d₆-DMSO) δ 8.40-7.83, 7.27,7.16-6.98, 6.83-6.64, 5.06-4.79, 4.62-4.18, 3.71-3.21, 2.98-2.78,2.75-2.58, 2.42-2.25, 2.22-2.13, 1.99-1.70.

Example 103

Synthesis ofmPEG11.5K-b-Poly-(d-Glu(OBn)_(3.5)-co-Glu(OBn)_(3.5))-b-Poly(Tyr(OBn)₂₅-co-d-Phe₁₅)-Ac

mPEG11.5KNH₂ (15 g, 1.3 mmol) prepared with the same method in Example 3with the exception of molecular weight, was weighed into a clean, ovendried, 1000 mL, two neck, round bottom flask and dissolved in toluene(300 mL) with heating and dried by azeotropic distillation. Afterdistillation to dryness, the polymer was left under vacuum for threehours. The flask was subsequently backfilled with N₂, re-evacuated underreduced pressure, and dry NMP:DCM (1:1) (450 mL) was introduced bycannula. Glu(OBn) NCA (1.2 g, 4.56 mmol) prepared with the same methodas Example 8, and d-Glu(OBn) NCA (1.2 g, 4.56 mmol) prepared with thesame method as Example 9, were added to the flask, and the reactionmixture was allowed to stir for 48 hours at ambient room temperatureunder nitrogen gas. Then, d-Phe NCA (2.88 g, 19.56 mmol) prepared withthe same method as Example 7 and Tyr (OBn) NCA (8.26 g, 32.60 mmol)prepared with the same method as Example 6, were added and the solutionwas allowed to stir at room temp for two hours and then heated to 35° C.for 48 hours at which point the reaction was complete (GPC, DMF/0.1%LiBr). The solution was cooled to room temperature and acetic anhydride(1.34 g, 13 mmol, 1.23 mL), N-methylmorpholine (NMM) (1.3 g, 13 mmol,1.43 mL) and dimethylaminopyridine (DMAP) (0.16 g, 1.3 mmole) wereadded. Stirring was continued for 16 hours at room temperature. Thepolymer was precipitated into diethyl ether:heptane 10:1 (2.5 L) andisolated by filtration, washed with fresh 100 mL portions of diethylether, and dried in vacuo to give the block copolymer as a fine, nearlycolorless powder (25 g, Yield=82.2%). ¹H NMR (d₆-DMSO) δ 8.38-7.80,7.42-7.18, 6.75, 5.02, 4.97-4.80, 4.66-4.16, 3.75-3.20, 3.02-2.80,2.76-2.56, 2.44-2.25, 2.00-1.59.

Example 104

Synthesis ofmPEG11.5K-b-Poly-(d-Glu(OBn)_(3.5)-co-Glu(OBn)_(3.5))-b-Poly(Tyr(OBn)₂₅-co-d-Phe₁₅)-Ac mPEG11.5KNH₂

(15 g, 1.3 mmol) prepared with the same method as Example 3 with theexception of molecular weight, was weighed into a clean, oven dried,1000 mL, two neck, round bottom flask and dissolved in toluene (300 mL)with heating and dried by azeotropic distillation. After distillation todryness, the polymer was left under vacuum for three hours. The flaskwas subsequently backfilled with N₂, re-evacuated under reducedpressure, and dry (NMP) and DCM (1:3 ratio) (450 mL) was introduced bycannula. Glu(OBn) NCA (1.2 g, 4.56 mmol) prepared with the same methodas Example 8, and d-Glu(OBn) NCA (1.2 g, 4.56 mmol) prepared with thesame method as Example 9, were added to the flask, and the reactionmixture was allowed to stir for 48 hours at ambient room temperatureunder nitrogen gas. Then, d-Phe NCA (2.88 g, 19.56 mmol) prepared withthe same method as Example 7, and Tyr (OBn) NCA (8.26 g, 32.60 mmol)prepared with the same method as Example 6, were added and the solutiondirectly and allowed to stir at room temperature for 2 hours and thenheated to 35° C. for 48 hours, at which point the reaction was complete(GPC, DMF/0.1% LiBr). The solution was cooled to room temperature andacetic anhydride (1.34 g, 13 mmol, 1.23 mL), N-methylmorpholine (NMM)(1.3 g, 13 mmol, 1.43 mL) and dimethylaminopyridine (DMAP) (0.16 g, 1.3mmole) were added. Stirring was continued for 16 hours at roomtemperature. The polymer was precipitated into diethyl ether:heptane10:1 (2.5 L) and isolated by filtration, washed with fresh 100 mLportions of diethyl ether, and dried in vacuo to give the blockcopolymer as a fine, nearly colorless powder (26 g, Yield=82.2%). ¹H NMR(d₆-DMSO) δ 8.46-7.85, 7.48-6.95, 6.84-6.61, 5.02, 4.97-4.79, 4.66-4.16,3.75-3.21, 3.00-2.79, 2.76-2.56, 2.43-2.25, 2.00-1.57.

Example 105

Synthesis of mPEG11.6K-b-Poly-(d-Glu(oBn)₅-co-Glu(oBn₅)-b-Poly(Tyr(OBn)₁₀-co-d-Leu₂₀-co-Asp(oTbu)₁₀-Ac

Using the general protocol from Example 73 and substituting appropriateNCA starting materials and using a 1:1 ratio of NMP:DCM resulted in thecrude polymer, this was precipitated with diethyl ether about 10volumes. After filtration and drying the title compound was collected asa colorless solid (30.5 g, Yield=87.1%). ¹H NMR (d6-DMSO) δ 8.39-7.94,7.41-7.17, 7.15-7.02, 6.82, 5.01, 4.60-4.16, 3.72-3.30, 2.70, 2.42-2.262.02-1.71, 1.33, 0.9-0.55.

Example 106

Synthesis of mPEG11.6K-b-Poly-(d-Glu(oBn)₅-co-Glu(oBn₅)-b-Poly(Tyr(OH)₁₀-co-d-Leu₂₀-co-Asp₁₀)-Ac

The triblock co-polymer from Example 105 was weighed (29 g, 1.38 mmol)into a clean 500 mL beaker and dissolved in triflouroacetic acid. Tothis solution pentamentyl-benzene (6.14 g, 41.4 mmol) was added andstirred with a magnetic stir-bar. At thirty mins post addition ofpentamethyl-benzene a precipitate was observed in solution. The reactionmixture was stirred for two hours and monitored by NMR for completeremoval of benzylic protecting groups on tyrosine and t-Butyl group onaspartate. After completion of this deprotection (5 Hrs) the solutionwas rotovapped to a thick paste, redissolved in methylene chloride andthen precipitated in cold diethyl ether and collected by filtration.This solid was washed three times with 100 mL portions of cold ether anddried in vacuo and characterized. (26 g, Yield=96.6%) ¹H NMR (d6-DMSO) δ9.09, 8.44-7.58, 7.35-6.89, 6.96, 6.58, 5.03, 4.62-4.16, 3.71-3.22,2.75-2.64, 2.40-2.26, 2.23-2.04, 0.92-0.54.

Example 107

Synthesis of mPEG11.6K-b-Poly-(d-Glu(NHOH)₅-co-Glu(NHOH)-b-Poly(Tyr(OH)₁₀-co-d-Leu₂₀-co-Asp₁₀)-Ac

Triblock ester from Example 106 was weighed (25 g, 1.38 mmol) into aclean 500 mL round bottom flask and the polymer was dissolved completelyin 200 mL of tetrahydrofuran. To this solution thirty equivalents ofhydroxylamine (1.9 mL, 0.028 mmol) and lithium hydroxide monohydrate(1.16 g, 27.6 mmol) was stirred under nitrogen at room temp over night.Completion of the reaction was verified by ¹H NMR. This solution wasmixed with 100 mL methanol and precipitated with diethyl ether (about 7volumes). This white solid was collected by filtration and washed withfresh diethyl ether. The collected solid was then dissolved in acetoneand and a catalytic amount of acetic acid was allowed to stir overnight.the solution was poured into a clean two liter beaker and diethyl etherwas slowly added to the solution with stirring. This white solid wascollected by filtration and then dried in vacuo. Yielded 22 g (92%). ¹HNMR (d6-DMSO)) δ 9.4-8.5, 8.40-7.71, 7.40-7.11, 6.93, 6.57, 5.10,4.53-3.99, 3.86-3.02, 2.99-2.87, 2.09-1.19, 1.6-1.2, 1.01-0.5.

Example 108

Synthesis of mPEG11.5K-b-Poly-(d-Glu(oBn)₅-co-Glu(oBn₅)-b-Poly(Tyr(OBn)₁₀-co-d-Phe₁₀-co-Asp(otBu)₁₀)-Ac

mPEG11.5KNH₂ (31 g, 2.7 mmol) prepared by the same method as Example 3except for the molecular weight, was weighed into a clean, oven dried,1000 mL, two neck, round bottom flask and dissolved in toluene (400 mL)with heating and dried by azeotropic distillation. After distillation todryness, the polymer was left under vacuum for three hours. The flaskwas subsequently backfilled with N₂, re-evacuated under reducedpressure, and a 1:2 ratio of Dimethylacetamide:methylene chloride wasintroduced by cannula and dissolved completely. Glu(OBn) NCA (3.4 g,12.9 mmol) prepared by the same method in Example 8, and d-Glu(OBn) NCA(3.4 g, 12.9 mmol) prepared by the same method as Example 9, wereweighed into a clean 500 mL round bottom flask and evacuated for 2 hoursbackfilled with N₂ and then dissolved in DMAC and cannulated into theflask containing PEG. This reaction mixture was allowed to stir for 14hours at ambient room temperature under nitrogen gas. Then, d-Phe NCA(5.15 g, 26.9 mmol) prepared by the same method as Example 7 and Tyr(OBn) NCA (7.99 g, 26.9 mmol) prepared by the same method as Example 6,were added in the same manner as mentioned above and the solution wasallowed to stir at room temp for two hours and then heated to 35° C. for26 hours at which point the reaction was complete (GPC, DMF/0.1% LiBr).The solution was cooled to room temperature and acetic anhydride (2.77g, 269 mmol, 2.46 mL), N-methylmorpholine (NMM) (2.69 g, 269 mmol, 1.43mL) and dimethylaminopyridine (DMAP) (0.33 g, 2.7 mmole) were added.Stirring was continued for 1 day at room temperature. The reactionsolution was rotovapped to remove the methylene chloride and then thepolymer was precipitated into isopropanol (3.5 L) and isolated byfiltration, washed with fresh 100 mL portions of isopropanol, and driedin vacuo to give the block copolymer as a nearly colorless powder (44.5g, Yield=83.1%). ¹H NMR (d₆-DMSO) δ 8.59-7.86, 7.45-7.25, 7.10, 6.79,5.12-4.79, 4.69-4.17, 3.84-3.23, 3.02-2.58, 2.40-2.23, 2.04-1.71, 1.33.

Example 109

Synthesis ofmPEG11.5K-b-Poly-(d-Glu(oBn)₅-co-Glu(oBn₅)-b-Poly-(Tyr(OH)₁₀-co-d-Phe₁₀-co-Asp(OH)₁₀)-Ac

Using the general method from Example 74 only adjusting scale, thispolymer was deprotected (30 g, 1.54 mmol). Once complete (3 Hrs.) thesolution was rotovapped to a thick paste and then redissolved in DCM andprecipitated in MTBE, collected by filtration, washed several times withfresh 100 mL portions of MTBE and dried in vacuo. This reaction yielded24 g of dry material (86.9%). ¹H NMR (d6-DMSO) δ 9.07, 8.50-7.80,7.40-7.28, 6.98, 6.62, 5.04, 4.69-4.17, 3.72-3.23, 3.02-2.76, 2.73-2.57,2.42-2.27, 2.23-1.59.

Example 110

Synthesis of mPEG11.5K-b-Poly-(d-Glu(NHOH)₅-co-Glu(NHOH)₅-b-Poly(Tyr(OH)₁₀-co-d-Phe₁₀-co-Asp(OH)₁₀)-Ac

The polymer from Example 109 (22 g, 1.2 mmol) was dissolved completelyin 200 mL of THF with heating. This solution was allowed to cool to roomtemp before 10M KOH solution was added (2 mL, 1.5 g, 10.8 mmol),followed by Hydroxyl amine (50% water solution, 6 mL, 3.6 g, 108 mmol)this solution was stirred at room temperature for 24 hours. Acetone 20mL and Acetic acid (2 mL) was added to this reaction solution andstirred 4 hours. The solution was rotovapped until nearly dry,redissolved in methylene chloride and precipitated in MTBE, collected byfiltration and dried in vacuo (20 g, Yield=94.9%). ¹H NMR (d₆-DMSO) δ8.61-7.90, 7.50-6.29, 5.38-5.01, 4.63-4.12, 3.78-3.22, 2.17, 2.11,1.81-1.63.

Example 111

Synthesis ofmPEG12K-b-Poly-(Asp(Ot-Bu)₁₀)-b-Poly-(d-Leu₂₀-co-Tyr(OBn)₂₀)-Ac

mPEG12KNH₂ from Example 3 (360 g, 30.0 mmol) was weighed into a clean,oven dried, 5000 mL, three neck, round bottom jacketed flask anddissolved in toluene (3000 mL) with heating in an oil bath at 55-60° C.and dried by azeotropic vacuum distillation. After about 30% of thetoluene was removed the distillation was stopped and Diflouroacetic acid(DFA) was added by syringe (2.26 mL, 0.036 mmol) to form the DFA salt.The solution was stirred for 30 minutes and then the azeotrope wasstarted again and dried completely. The polymer salt was left undervacuum overnight. The flask was subsequently backfilled with N₂,re-evacuated under reduced pressure, and dry N-methylpyrrolidone (NMP)(3500 mL) was introduced by cannula. The mixture was briefly heated to40° C. to expedite dissolution and then cooled to 25° C. Asp(OtBu) NCA(64.56 g, 300 mmol) was weighed into a clean 1 L, 2 neck RBF andevacuated for an hour before freshly distilled NMP was cannulated intothe flask and completely dissolved the NCA. This solution was thencannulated into the PEG flask and allowed to stir at room temperaturefor 48 hours under nitrogen gas. Then, d-Leu NCA (94.30 g, 600 mmol) andTyr (OBn) NCA (178.39 g, 600 mmol) were added to the solution by thesame method as described above and the resultant solution was allowed tostir at 35° C. for 48 hours at which point the reaction was deemedcomplete (GPC, DMF/0.1% LiBr). The solution was cooled to roomtemperature and acetic anhydride (45.9 g, 0.45 mol, 42.5 mL), pyridine(59.3 g, 0.75 mol, 60.7 mL) and dimethylaminopyridine (DMAP) (0.37 g,3.0 mmole) were added. Stirring was continued for 1 day at roomtemperature. The polymer was precipitated into 5 volumes of diethylether (15 L) and isolated by filtration, washed with fresh 300 mLportions of diethyl ether, and dried in vacuo to give the blockcopolymer as a fine, off white powder (434.9 g, Yield=69.0%). ¹H NMR(d₆-DMSO) δ 8.50-7.90, 7.60-7.30, 7.25-6.77, 5.10-4.85, 4.65-4.10,3.72-3.25, 3.05-2.45, 2.44-1.60, 1.40-1.25, 0.90-0.50.

Example 112

Synthesis of mPEG12K-b-Poly-(Asp(OH)₁₀)-b-Poly-(d-Leu₂₀-co-Tyr(OH)₂₀)-Ac

mPEG12K-b-Poly-(Asp(Ot-Bu)₁₀)-b-Poly-(d-Leu₂₀-co-Tyr(OBn)₂₀)-Ac fromExample 111 (314.5 g, 14.9 mmol) and pentamethylbenzene (141.4 g, 0.954mole) were dissolved into 2.2 L of trifluoroacetic acid (TFA). Thereaction was rapidly stirred for 14 hours at ambient room temperature.The TFA was removed on a rotary evaporator with the water bathtemperature not exceeding 35° C. The resultant putty-like solid wasdissolved in 1.4 L of dichloromethane, transferred to a 12 L tub, andprecipitated by slow addition of 5.6 L of diethyl ether using rapidmechanical stirring. The resultant slurry was stirred for 30 minutes,solids were collected by filtration, washed with 2×1 L portions of freshdiethyl ether, and vacuum dried. The solid was redissolved in 900 mL ofdichloromethane and precipitated by addition of 10 L of diethyl ether.Filtration and vacuum drying afforded the product as a colorless, fluffysolid (254.4 g, Yield=91.3%). ¹H NMR (d₆-DMSO) δ 12.4, 9.09, 8.50-7.80,7.05-6.45, 4.65-4.0, 3.85-3.1, 3.03-2.45, 2.44-1.63, 1.58-0.95,0.90-0.50.

Example 113

Formulation of Daunorubicin—

Triblock copolymer from Example 35[mPEG12K-b-Poly-[d-Glu(NHOH)₅-co-Glu(NHOH)₅]-b-Poly-(Tyr(OH)₃₀-co-d-Phe₁₀)-Ac](330 mg) was dissolved in water at 1.65 mg/mL by stirring at ˜50° C. for10 minutes. The solution was allowed to cool and the pH was adjusted to7.0 with 0.1 N NaOH. Daunorubicin feed rate for the formulation was 10%of the polymer weight. An organic solution (20% methanol, 80%dichloromethane) was used to dissolve 33 mg daunorubicin at 8.25 mg/mLby placing the solution in a sonicating water bath followed by heatingand vortexing, and repeating until a clear, red solution persisted. Theorganic solution was allowed to cool to room temperature then 17 μL oftriethylamine was added. The organic solution was then added to thepolymer solution while shear mixing at 10,000 RPM for ˜1 minute. Theresulting emulsion, which was a turbid, red solution, was allowed tostir in a fume hood over night. As the organic solvent evaporated thesolution became less turbid and more red in color. The next day thesolution was filtered through a 0.22 micron, dead end filter. Atangential flow filtration apparatus equipped with a 10 kD cutoff filterwas used to concentrate the sample from 200 mL to approximately 50 mL.The formulation was then frozen at −70° C. and lyophilized. Formulationof daunorubicin resulted in an 88% yield. Weight loading was determinedby comparing a standard curve of daunorubicin to a known concentrationof formulation by HPLC analysis. Daunorubicin was dissolved in methanolin a range from 40 μg/mL to 200 μg/mL, and the formulation was dissolvedat 2 mg/mL in methanol. The amount of daunorubicin in the formulation isthen converted to % based on the known quantity of formulation used(i.e. 2 mg/mL). This formulation demonstrated a weight loading of 7.8%from a 10% feed; representing a 69% efficient process. Particle sizeanalysis of the uncrosslinked formulation by dynamic light scatteringresulted in average diameter of 75 nm. Encapsulation of daunorubicin wasverified by dialysis of the uncrosslinked formulation above the criticalmicelle concentration (CMC) at 20 mg/mL, and below the CMC at 0.2 mg/mL.As shown in FIG. 5, the formulation dialyzed above the CMC resulted inapproximately 88% retention of daunorubicin while dialysis below the CMCresulted in approximately 15% retention of daunorubicin. This resultshows that the daunorubicin is effectively encapsulated in the micelleat high concentrations (above the CMC) and that the micelle falls apartwhen diluted below the CMC.

Example 114

Crosslinking of Daunorubicin Loaded Micelles

The daunorubicin loaded micelles of Example 113 were in water at 20mg/mL with 0.1, 0.25, 0.5, 0.75, 1, 2.5, 5, 7.5 or 10 mM iron (III)chloride for approximately 16 hours. Each of the nine separate sampleswas diluted to 0.2 mg/mL and dialyzed for 6 hours against phosphatebuffer pH 8 to determine the extent of crosslinking. The result of thisexperiment is shown in FIG. 6. This result demonstrates thatdaunorubicin loaded micelles are stable to dilution (crosslinked) whentreated with iron (III) chloride, with the best results obtained withconcentrations above 5 mM of iron (III) chloride.

Example 115

Optimization of Crosslinking Time

The daunorubicin loaded micelles of Example 113 were in 10 mM iron (III)chloride at 20 mg/mL. Aliquots of the sample were taken at 5 minutes, 30minutes, 1 hour, 2 hours, 4 hours and 16 hours, along with anuncrosslinked sample with no iron at 5 minutes, diluted to 0.2 mg/mL anddialyzed against 10 mM phosphate buffer pH 8 for 6 hours. The %daunorubicin remaining post dialysis for the time-dependent crosslinkingis shown in FIG. 7. Based on FIG. 3 the crosslinking of the sampleoccurred rapidly, with nearly 70% retention of the daunorubicinremaining after just 5 minutes incubation of the sample with the iron(III) chloride solution prior to dilution below the CMC.

Example 116

Optimization of Crosslinking pH

The daunorubicin loaded micelles of Example 113 were in 10 mM iron (III)chloride at 20 mg/mL then aliquots of this solution adjusted to pH 3, 4,5, 6, 7, 7.4 and 8 with dilute sodium hydroxide and stirred for 10minutes. Each sample was then diluted to 0.2 mg/mL and dialyzed against10 mM phosphate buffer pH 8 for 6 hours. The % daunorubicin remainingpost dialysis against 10 mM phosphate buffer pH 8 is shown in FIG. 8.This result demonstrated that the optimal pH for crosslinking is 7.4.

Example 117

pH Dependent Release of Daunorubicin from Crosslinked Micelles

The daunorubicin loaded micelles of Example 113 were in 10 mM iron (III)chloride at 20 mg/mL then adjusted to pH 7.4 with dilute sodiumhydroxide and stirred for 10 minutes. This sample was then diluted to0.2 mg/mL and dialyzed against 10 mM phosphate buffer at pH 3, 4, 5, 6,7, 7.4 and 8 for 6 hours. The % daunorubicin remaining post dialysisagainst 10 mM phosphate buffer as a function of pH is shown in FIG. 9.This result demonstrates a pH dependent release of drug from acrosslinked micelle.

Example 118

Salt Dependent Release of Daunorubicin from Crosslinked Micelles

The daunorubicin loaded micelles of Example 113 were in 10 mM iron (III)chloride at 20 mg/mL then adjusted to pH 7.4 with dilute sodiumhydroxide and stirred for 10 minutes. Each sample was then diluted to0.2 mg/mL and dialyzed against 10 mM phosphate buffer at pH 8 withconcentrations ranging from 0 to 500 mM NaCl for 6 hours. The %daunorubicin remaining post dialysis against 10 mM phosphate buffer as afunction of salt concentration is shown in FIG. 10. This resultdemonstrates a salt dependent release of drug from a crosslinkedmicelle.

Example 119

Encapsulation of Aminopterin—

Triblock copolymer from Example 62[mPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(d-Leu₁₅-co-Asp(OH)₅-co-Tyr(OH)₂₀)-Ac](800 mg) was dissolved in water at 2 mg/mL by stirring at ˜50° C. for 30minutes. The solution was allowed to cool and the pH was adjusted to 7.0with 0.1 N NaOH. Aminopterin feed for the formulation was 4% of thepolymer weight. An organic solution (20% methanol, 80% dichloromethane,15 mg/mL para-toluenesufonic acid) was used to dissolve 32 mgaminopterin at 3.2 mg/mL by placing the solution in a sonicating waterbath followed by heating and vortexing, and repeating until a clear,yellow solution persisted. Once the organic solution cooled it was addedto the polymer solution while shear mixing at 10,000 RPM for ˜1 minute.The resulting emulsion, which was a turbid, yellow solution, was allowedto stir in a fume hood over night. As the organic solvent evaporated thesolution became less turbid and more yellow in color. The next day thesolution was pH adjusted to 7.0 with NaOH and filtered through a 0.22micron, dead end filter. A tangential flow filtration apparatus equippedwith a 10 kD cutoff filter was used for diafiltration with a three-foldbuffer exchange to remove unencapsulated aminopterin and trace solvents.The formulation was then frozen at −70° C. and lyophilized. Formulationof aminopterin with the triblock copolymer resulted in an 85% yield ofproduct. Weight loading was determined by comparing a standard curve ofaminopterin to a known concentration of formulation by HPLC analysis.Aminopterin was dissolved in HPLC mobile phase (60% acetonitrile, 40% 10mM phosphate buffer pH 8) in a range from 40 μg/mL to 200 μg/mL, and theformulation was dissolved at 5 mg/mL in HPLC mobile phase. The amount ofaminopterin in the formulation is then converted to % based on the knownquantity of formulation used (i.e. 5 mg/mL). The aminopterin-loadedmicelle was found to have a loading of 2.5% weight loading from a 4%feed, resulting in a 53% efficient process. Particle size of theuncrosslinked formulation demonstrated a single distribution averageparticle size of approximately 70 nm, as shown in FIG. 11.

Example 120

Verification of Aminopterin Encapsulation

The aminopterin loaded micelles from Example 119 was dissolved at 20mg/mL in 10 mM phosphate buffer pH 8. The uncrosslinked formulation wasalso diluted below the CMC (0.2 mg/mL) and dialyzed against 10 mMphosphate buffer pH 8 for six hours. The histogram shown in FIG. 12demonstrates the stability of the uncrosslinked formulation at 20 mg/mL,with greater than 75% of the aminopterin remaining inside the dialysisbag over 6 hours. However, when diluted to 0.2 mg/mL, less than 10% ofthe aminopterin was left in the dialysis bag after 6 hours. This resultshows that the aminopterin is effectively encapsulated in the micelle athigh concentrations (above the CMC) and that the micelle falls apartwhen diluted below the CMC.

Example 121

pH Dependent Release of Aminopterin from Crosslinked Micelles

The aminopterin loaded micelles from Example 119 was dissolved at 20mg/mL in 10 mM iron (III) chloride and stirred for 10 minutes. Thissample was then diluted to 0.2 mg/mL and dialyzed against 10 mMphosphate buffer at pH 3, 4, 5, 6, 7, 7.4 and 8 for 6 hours. The %aminopterin remaining post dialysis against 10 mM phosphate buffer as afunction of pH is shown in FIG. 13. This result demonstrates a pHdependent release of aminopterin from a crosslinked micelle.

Example 122

Cytotoxicity of Aminopterin Loaded Crosslinked Micelles

The aminopterin loaded micelles from Example 119 and Example 121 weretested for cytotoxicity compared to free aminopterin and the crosslinkedand uncrosslinked non drug-loaded micelle formulations (from polymer ofExample 18) against A549 lung, OVCAR3 ovarian, PANC-1 (folate receptor+)pancreatic and BxPC3 (folate receptor−) pancreatic cancer cell lines.The cytotoxicity profiles for each treatment for each cell line in FIG.14 (A549 Lung), FIG. 15 (OVCAR3 Ovarian), FIG. 16 (PANC-1 Pancreatic),and FIG. 17 (BxPC3 pancreatic). Aminopterin inhibited cell viability by50% (IC₅₀) in the low nanomolar range (˜7-25 nM) in A549 and PANC-1cells, however no IC₅₀ was obtained for OVCAR3 or BxPC3 cells. Likewise,the uncrosslinked and crosslinked formulations demonstrated IC₅₀ valuesin the low nanomolar range (˜20-70 nM) for A549 and PANC-1 cells withoutreaching 50% inhibition in OVCAR3 or BxPC3 cells. Treatment with bothuncrosslinked and crosslinked non drug-loaded micelles was welltolerated, with greater than 80% viability for all cells tested.

Example 123

Encapsulation of Berberine—

Triblock copolymer from Example 62[mPEG12K-b-Poly-(d-Glu(OBn)₅-co-Glu(OBn)₅)-b-Poly(d-Leu₁₅-co-Asp(OH)₅-co-Tyr(OH)₂₀)-Ac](300 mg) was dissolved in water at 2 mg/mL by stirring at ˜50° C. for 10minutes. The solution was allowed to cool and the pH was adjusted to 7.0with 0.1 N NaOH. Berberine feed rate for the formulation was 5% of thepolymer weight. An organic solution (20% methanol, 80% dichloromethane)was used to dissolve 15 mg berberine at 6 mg/mL by vortexing until aclear, yellow solution persisted. The organic solution was then added tothe polymer solution while shear mixing at 10,000 RPM for ˜1 minute. Theresulting emulsion, which was a turbid, yellow solution, was allowed tostir in a fume hood over night. As the organic solvent evaporated thesolution became less turbid and more yellow in color. The next day thesolution was filtered through a 0.22 micron, dead end filter. Atangential flow filtration apparatus equipped with a 10 kD cutoff filterwas used to concentrate the sample from 200 mL to approximately 50 mL.The formulation was then frozen at −70° C. and lyophilized. Weightloading was determined by comparing a standard curve of berberine to aknown concentration of formulation by HPLC analysis. Berberine wasdissolved in methanol in a range from 40 μg/mL to 200 μg/mL, and theformulation was dissolved at 5 mg/mL in methanol. The amount ofberberine in the formulation was then converted to % based on the knownquantity of formulation used (i.e. 5 mg/mL). Weight loading of theberberine formulation was 4% from a 5% feed, as determined by HPLCanalysis of the formulation compared to a standard curve of the freedrug. Encapsulation efficiency of the formulation was 72%. Particle sizeanalysis by dynamic light scattering resulted in an average particlesize of 72.5 nm for the uncrosslinked sample. Encapsulation dialysisresulted in 53% retention, demonstrating that berberine is effectivelyencapsulated in the micelle.

Example 124

Crosslinking of the Berberine Loaded Micelle—

The lyophilized uncrosslinked powder from Example 123 was reconstitutedin water at 20 mg/mL. Iron (III) chloride was added to the solution fora final concentration of 5 mM, and stirred for ˜30 minutes. Theformulation was then frozen at −70° C. and lyophilized. To verifycrosslinking the uncrosslinked and crosslinked samples were diluted to0.2 mg/mL and dialyzed for 6 hours. The uncrosslinked micelle showed 5%of the berberine retained, while the crosslinked sample showed 43%berberine remaining. This result demonstrates that the berberine micelleis stabilized by the addition of iron.

Example 125

Encapsulation of Paclitaxel—

Triblock copolymer from Example 38[mPEG12K-b-Poly-[d-Glu(NHOH)₅-co-Glu(NHOH)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac](300 mg) was dissolved in water at 2 mg/mL by stirring at ˜50° C. for 10minutes. The solution was allowed to cool and the pH was adjusted to 7.0with 0.1 N NaOH. Paclitaxel feed rate for the formulation was 1% of thepolymer weight. An organic solution (20% methanol, 80% dichloromethane)was used to dissolve 3 mg paclitaxel at 3 mg/mL by vortexing until aclear, colorless solution persisted. The organic solution was then addedto the polymer solution while shear mixing at 10,000 RPM for ˜1 minute.The resulting emulsion was allowed to stir in a fume hood over night.The next day the solution was filtered through a 0.22 micron, dead endfilter. The formulation was then frozen at −70° C. and lyophilized.Weight loading of the paclitaxel formulation was 0.78% from a 1% feed,as determined by HPLC analysis of the formulation compared to a standardcurve of the free drug. Particle size analysis by dynamic lightscattering resulted in an average particle size of 45.7 nm for theuncrosslinked sample. Encapsulation verification dialysis above thecritical micelle concentration (20 mg/mL) resulted in 52% retention ofthe paclitaxel post dialysis.

Example 126

Encapsulation of SN-38—

Triblock copolymer from Example 38[mPEG12K-b-Poly-[d-Glu(NHOH)₅-co-Glu(NHOH)₅]-b-Poly-(Tyr(OH)₂₅-co-d-Phe₁₅)-Ac](1 g) was dissolved at 5 mg/mL in water by stirring at ˜50° C. for 10minutes. Sucrose (1 g) was then added to the polymer solution andstirred until fully dissolved. The solution was allowed to cool to roomtemperature and pH adjusted to 6.0 with 0.1 N NaOH. SN-38 feed for theformulation was 3% of the polymer weight. DMSO was used to dissolve 30mg SN-38 at 80 mg/mL by heating, vortexing and placing the solution in asonicating water bath until a clear, yellow solution persisted. Theorganic solution was allowed to cool to room temperature and was thenadded to the polymer solution while shear mixing at 10,000 RPM for ˜1minute. The resulting emulsion, which was a turbid, light yellowsolution, was then then transferred to the feed chamber of amicrofluidizer. The solution was processed with a single pass through aMicrofluidics M110Y microfluidizer. The microfluidizer outlet stream wascooled with an ice water bath. The solution was then filtered through a0.22 micron dead-end filter, and the resulting solution was thensubjected to ultrafiltration with a Spectrum Labs KrosFlo tangentialflow filtration system and a 10 kDa diafiltration membrane. The solutionwas concentrated from 200 mL to ˜50 mL, then 150 mL of water with 3mg/mL sucrose was added and concentrated back down to ˜50 mL. Theultrafiltration was repeated until a total of 4-times the originalvolume of buffer was exchanged (800 mL). The resulting solution was thenfrozen at −70° C. and lyophilized.

Example 127

Crosslinking of SN-38 Micelle—

SN-38 micelles from Example 126 were dissolved at 20 mg/mL in aqueous 10mM FeCl₃. The pH was then adjusted to 6.8 with dilute NaOH. The solutionwas stirred for 1 h at room temperature then lyophilized. Thiscrosslinked, SN-38 loaded micelle was isolated as brownish powder with aweight loading of 1.75%, representing a 81.4% efficient process.Particle size analysis by dynamic light scattering resulted in anaverage diameter of 70 nm.

Example 128

Preparation and Crosslinking SN-38 Micelles—

Formulations were done with the following polymers:128A=mPEG12k-b-p[Glu(NHOH)₂]-b-p[Phe₁₅-co-Tyr₂₅]-Ac, from Example 81;128B=mPEG12k-b-p[Glu(NHOH)₇]-b-p[Phe₁₅-co-Tyr₂₅]-Ac from Example 56;128C=mPEG12k-b-p[Glu(NHOH)₁₀]-b-p[Phe₁₅-co-Tyr₂₅]-Ac, from Example 38;128D=mPEG12k-b-p[Glu(NHOH)₂₀]-b-p[Phe₁₅-co-Tyr₂₅]-Ac, from Example 98;and 128E=mPEG12k-b-p[Asp₁₀]-b-p[Leu₂₀-co-Tyr₂₀]-Ac from Example 112.Triblock copolymer (1 g) was dissolved at 5 mg/mL in water by stirringat ˜40° C. for 30 minutes. 1 g of sucrose was then added to the polymersolution and stirred until fully dissolved. The solution was allowed tocool to room temperature and pH adjusted to 6.0 with NaOH. SN38 feedrate for the formulation was 5% of the polymer weight. DMSO was used todissolve 50 mg SN-38 at 80 mg/mL by heating, vortexing and placing thesolution in a sonicating water bath until a clear, yellow solutionpersisted. The organic solution was allowed to cool to room temperatureand was then added to the polymer solution while shear mixing at 10,000RPM for ˜1 minute. The resulting emulsion, which was a turbid, lightyellow solution, was then then transferred to the feed chamber of amicrofluidizer. The solution was processed with a single pass throughthe microfluidizer. The microfluidizer outlet stream was cooled with anice water bath. The solution was then filtered through a 0.22 microndead-end filter, and the resulting solution was then subjected toultrafiltration with a Spectrum Labs KrosFlo tangential flow filtrationsystem and a 10 kDa diafiltration membrane. The solution wasconcentrated from 200 mL to ˜50 mL, then 150 mL of water with 5 mg/mLsucrose was added and concentrated back down to ˜50 mL. Theultrafiltration was repeated until a total of 4-times the originalvolume of buffer was exchanged (800 mL). Iron (III) Chloride was thenadded to the formulation for a final concentration of 10 mM. The pH ofthe solution was then adjusted to 6.0 with NaOH and stirred at roomtemperature for 4 hours. One volume of buffer containing sucrose at 20mg/mL was then added to the solution, and then concentrated back down toapproximately 20 mg/mL polymer concentration. The solution was thenfrozen at −40 degrees Celsius and lyophilized. Formulations of SN-38with triblock copolymers resulted in an average yield of 85% of productwith a weight loading of 3.5%. Actual weight loadings: A=3.4%, B=3.2%,C=3.6%, D=3.6%, E=3.2%. Particle size analysis by dynamic lightscattering resulted in an average diameter of 90 nm. Actual particlesizes: A=84 nm B=88 nm, C=89 nm, D=110 nm, E=91 nm.

Example 129

Parmacokinetics of Crosslinked SN-38 Micelles—

Sprague-Dawly rats surgically modified with jugular vein catheters werepurchased from Harlan Laboratories, Dublin, Va. SN-38 crosslinkedformulations (From Example 128C and 128E) were dissolved in water with150 mM NaCl for a final concentration of 10 mg SN-38 per kg animal bodyweight for 2 mL bolus injection via JVC over approximately 1 minute,followed by a flush of approximately 250 heparinized saline. Time pointsfor blood collection following test article administration were asfollowed: 1, 5, 15 minutes, 1, 4, and 24 hours. Approximately 250 μL ofblood per time point was collected by JVC into K3-EDTA blood collectiontubes followed by a flush of approximately 200 μL heparinized saline.Blood was then centrifuged at 2000 RPM for 5 minutes to isolate plasma.Plasma was then collected and snap frozen until processed for HPLCanalysis. Samples were prepared for analysis by first thawing the plasmasamples at room temperature. 50 μL plasma was added to a 2 mL eppendorftube 150 μL of extraction solution (0.1% phosphoric acid in methanol, 5μg/mL camptothecin internal standard). Samples were then vortexed for 10minutes and centrifuged for 10 minutes at 13,000 RPM. Supernatant wasthen transferred into HPLC vials then analyzed by HPLC. Quantitation ofSN-38 was determined using a standard curve of SN-38 formulation in ratplasma compared to samples collected from rats at each time point. Theresults of this experiment are shown in FIG. 18. The CMax of SN-38 inthe plasma from IT-141 (NHOH; 128C) was 304.5 μg/mL, determined 1 minutepost administration. The exposure of SN-38 to the plasma compartmentdelivered by the hyrdoxyamic acid formulation was 111.5 μg*h/mL. Theexposure of SN-38 to the plasma compartment from IT-141 (Asp; 127E) was31.6 μg*h/mL, with a CMax of 156.0 μg/mL.

Example 130

Determination of Optimal Crosslinking Block Length Determined by RatPharmacokinetics

Using the procedure of Example 129, Formulations of Examples 128A, 128B,and 128D were administered to rats at 10 mg/kg. The CMax of SN-38 in theplasma from Example 128D (NHOH-20) was 292.9 μg/mL, determined 1 minutepost administration. The exposure of SN-38 to the plasma compartment asdetermined by the area under the concentration versus time curvedelivered by the formulation was 85.7 μg*h/mL. The exposure of SN-38 tothe plasma compartment from Example 128B(NHOH-7) was 71.3 μg*h/mL, witha CMax of 256.9 μg/mL determined at 1 minute post administration. TheCMax of SN-38 in the plasma from Example 128A (NHOH-2) was 267.7 μg/mL,determined 1 minute post administration. The exposure of SN38 to theplasma compartment as determined by the area under the concentrationversus time curve delivered by the formulation was 41.8 μg*h/mL. Theresults are shown in FIG. 19. It was determined that Example 128Cdemonstrated the optimal crosslinking results.

Example 131

Preparation of Daunorubicin Loaded Micelles

Triblock copolymer from Example 112 (Aspartic acid core block) and water(2 L) was added to a 4 L beaker and stirred until a homogeneous solutionwas present. Daunorubicin hydrochloride (301 mg) was suspended in 4:1dichloromethane:methanol (60 mL), followed by the addition oftriethylamine (82 uL). The resulting daunorubicin suspension was addeddropwise to the rapidly stirring aqueous solution. The resultingsolution was covered with foil and allowed to stir for an additionaleight hours. The solution was filtered through a 0.22 μm filter and thenlyophilized to give 2.95 g (89% yield) as a red powder. A portion ofthis material was dissolved at 25 mg/mL polymer concentration in 20 mMTris, pH 7.5 supplemented with 5 mM FeCl₃. Once a homogeneous solutionwas present, the pH was adjusted to 8.0 with 1 N NaOH, then stirredovernight. The solution was frozen and lyophilized to give a dark redpowder.

Example 132

Preparation of Aminopterin Micelles

Triblock copolymer from Example 30mPEG12k-b-p[Glu(NHOH)10]-b-p[Asp5-co-Leu15-co-Tyr20]-Ac (800 mg) wasdissolved in water at 2 mg/mL by stirring at ˜40° C. for 30 minutes. Thesolution was allowed to cool and the pH was adjusted to 7.0 with NaOH.Aminopterin feed rate for the formulation was 4% of the polymer weight.An organic solution (20% methanol, 80% dichloromethane, 25 mg/mLpara-toluenesufonic acid) was used to dissolve 32 mg aminopterin at 3.2mg/mL by placing the solution in a sonicating water bath followed byheating and vortexing, and repeating until a clear, yellow solutionpersisted. Once the organic solution cooled it was added to the polymersolution while shear mixing at 10,000 RPM for ˜1 minute. The resultingemulsion, which was a turbid, yellow solution, was allowed to stir in afume hood over night. As the organic solvent evaporated the solutionbecame less turbid and more yellow in color. The next day the solutionwas pH adjusted to 7.0 with NaOH and filtered through a 0.22 microndead-end filter, and the resulting solution was then subjected toultrafiltration with a Spectrum Labs KrosFlo tangential flow filtrationsystem and a 10 kDa diafiltration membrane. The solution wasconcentrated from 2 mg/mL polymer concentration to approximately 20mg/mL polymer concentration, and Iron (III) Chloride was added to theformulation for a final concentration of 10 mM. The pH of the solutionwas then adjusted to 7.0 with NaOH and stirred at room temperature for 4hours. The solution was then adjusted to 5 mg/mL polymer concentrationwith water, and concentrated to approximately 20 mg/mL byultrafiltration. The solution was then frozen at −40 degrees Celsius andlyophilized. Formulation of aminopterin with the triblock copolymerresulted in an 85% yield of product with a 2.5% weight loading from a 4%feed, resulting in a 53% efficient process. Particle size of theuncrosslinked and crosslinked formulations demonstrated a singledistribution average particle size of approximately 70 nm.

Example 133

Preparation of Cabizataxel Micelles

Triblock copolymer from Example 38mPEG12k-b-p[Glu(NHOH)10]-b-p[Phe15-co-Tyr25]-Ac (300 mg) was dissolvedin water at 2 mg/mL by stirring at ˜40 degrees Celsius for 30 minutes.The solution was allowed to cool and the pH was adjusted to 7.0 withNaOH. Cabazitaxel feed rate for the formulation was 1.5% of the polymerweight. An organic solution (20% methanol, 80% dichloromethane) was usedto dissolve 4.5 mg cabazitaxel at 2 mg/mL by vortexing until a clear,colorless solution persisted. The organic solution was then added to thepolymer solution while shear mixing at 10,000 RPM for ˜1 minute. Theresulting emulsion was allowed to stir in a fume hood over night. Thenext day the solution was filtered through a 0.22 micron dead endfilter, and the resulting solution was then subjected to ultrafiltrationwith a Spectrum Labs KrosFlo tangential flow filtration system and a 10kDa diafiltration membrane. The solution was concentrated from 2 mg/mLpolymer concentration to approximately 20 mg/mL polymer concentration,and Iron (III) Chloride was added to the formulation for a finalconcentration of 10 mM. The pH of the solution was then adjusted to 7.0with NaOH and stirred at room temperature for 4 hours. The solution wasthen adjusted to 5 mg/mL polymer concentration with water, andconcentrated to approximately 20 mg/mL by ultrafiltration. The solutionwas then frozen at −40 degrees Celsius and lyophilized. Weight loadingfor the cabazitaxel formulation was 1% from a 1.5% feed. Particle sizeof the formulation was 62 nm in diameter. Encapsulation dialysis of theuncrosslinked formulation resulted 68% retention above the CMC at 20mg/mL, and 72% retention when the crosslinked formulation was diluted to0.2 mg/mL. FIG. 20 shows the results of the pH dependent crosslinkingdialysis for crosslinked Cabizataxel micelles.

Example 134

Preparation of Epothilone D Micelles

Triblock copolymer from Example 98mPEG12k-b-p[Glu(NHOH)20]-b-p[Phe15-co-Tyr25]-Ac (300 mg) was dissolvedin water at 2 mg/mL by stirring at ˜40 degrees Celsius for 30 minutes.The solution was allowed to cool and the pH was adjusted to 7.0 withNaOH. Epothilone D feed rate for the formulation was 2% of the polymerweight. An organic solution (20% methanol, 80% dichloromethane) was usedto dissolve 6 mg epothilone D at 2 mg/mL by vortexing until a clear,colorless solution persisted. The organic solution was then added to thepolymer solution while shear mixing at 10,000 RPM for ˜1 minute. Theresulting emulsion was allowed to stir in a fume hood over night. Thenext day the solution was filtered through a 0.22 micron dead endfilter, and the resulting solution was then subjected to ultrafiltrationwith a Spectrum Labs KrosFlo tangential flow filtration system and a 10kDa diafiltration membrane. The solution was concentrated from 2 mg/mLpolymer concentration to approximately 20 mg/mL polymer concentration,and Iron (III) Chloride was added to the formulation for a finalconcentration of 10 mM. The pH of the solution was then adjusted to 6.0with NaOH and stirred at room temperature for 4 hours. The solution wasthen adjusted to 5 mg/mL polymer concentration with water, andconcentrated to approximately 20 mg/mL by ultrafiltration. The solutionwas then frozen at −40 degrees Celsius and lyophilized. This processresulted in a 71% efficient process with a weight loading of 1.5% from a2% feed and an overall yield of 94%. The particle size of theformulation was 82 nm in diameter. Encapsulation dialysis of theuncrosslinked formulation resulted in 88% retention of epothilone D over6 hours at 20 mg/mL, while dilution to 0.2 mg/mL resulted in 10%retention of the drug over 6 hours.

Example 135

Preparation of Berberine Micelles

Triblock copolymer from Example 98mPEG12k-b-p[Glu(NHOH)10]-b-p[Asp5-co-Leu15-co-Tyr20]-Ac (300 mg) wasdissolved in water at 2 mg/mL by stirring at ˜40 degrees Celsius for 30minutes. The solution was allowed to cool and the pH was adjusted to 7.0with 0.1 N NaOH. Berberine feed rate for the formulation was 5% of thepolymer weight. An organic solution (20% methanol, 80% dichloromethane)was used to dissolve 15 mg berberine at 6 mg/mL by vortexing until aclear, yellow solution persisted. The organic solution was then added tothe polymer solution while shear mixing at 10,000 RPM for ˜1 minute. Theresulting emulsion, which was a turbid, yellow solution, was allowed tostir in a fume hood over night. As the organic solvent evaporated thesolution became less turbid and more yellow in color. The next day thesolution was filtered through a 0.22 micron dead-end filter, and theresulting solution was then subjected to ultrafiltration with a SpectrumLabs KrosFlo tangential flow filtration system and a 10 kDadiafiltration membrane. The solution was concentrated from 2 mg/mLpolymer concentration to approximately 20 mg/mL polymer concentration,and Iron (III) Chloride was added to the formulation for a finalconcentration of 10 mM. The pH of the solution was then adjusted to 7.0with NaOH and stirred at room temperature for 4 hours. The solution wasthen adjusted to 5 mg/mL polymer concentration with water, andconcentrated to approximately 20 mg/mL by ultrafiltration. The solutionwas then frozen at −40 degrees Celsius and lyophilized. Weight loadingof the berberine formulation was 4% from a 5% feed, as determined byHPLC analysis of the formulation compared to a standard curve of thefree drug. Encapsulation efficiency of the formulation was 72%. Particlesize analysis by dynamic light scattering resulted in an averageparticle size of 66.7 nm in diameter for the crosslinked sample, and72.5 nm for the uncrosslinked sample.

Example 136

Preparation of Vinorelbine Micelles

Triblock copolymer from Example 38 (300 mg) was dissolved in water at 2mg/mL by stirring at ˜40 degrees Celsius for 30 minutes. The solutionwas allowed to cool and the pH was adjusted to 7.0 with 0.1 N NaOH.Vinorelbine feed rate for the formulation was 5% of the polymer weight.An organic solution (20% methanol, 80% dichloromethane) was used todissolve 15 mg vinorelbine at 6 mg/mL by vortexing until a clear,colorless solution persisted. The organic solution was then added to thepolymer solution while shear mixing at 10,000 RPM for ˜1 minute. Theresulting emulsion, which was a turbid solution, was allowed to stir ina fume hood over night. As the organic solvent evaporated the solutionbecame less turbid and colorless. The next day the solution was filteredthrough a 0.22 micron dead-end filter, and the resulting solution wasthen subjected to ultrafiltration with a Spectrum Labs KrosFlotangential flow filtration system and a 10 kDa diafiltration membrane.The solution was concentrated from 2 mg/mL polymer concentration toapproximately 20 mg/mL polymer concentration, and Iron (III) Chloridewas added to the formulation for a final concentration of 10 mM. The pHof the solution was then adjusted to 7.0 with NaOH and stirred at roomtemperature for 4 hours. The solution was then adjusted to 5 mg/mLpolymer concentration with water, and concentrated to approximately 20mg/mL by ultrafiltration. The solution was then frozen at −40 degreesCelsius and lyophilized.

Example 137

Preparation of Everolimus Micelles

Triblock copolymer from Example 38 (300 mg) was dissolved in water at 2mg/mL by stirring at ˜40 degrees Celsius for 30 minutes. The solutionwas allowed to cool and the pH was adjusted to 7.0 with 0.1 N NaOH.Everolimus feed rate for the formulation was 5% of the polymer weight.An organic solution (20% methanol, 80% dichloromethane) was used todissolve 15 mg everolmus at 6 mg/mL by vortexing until a clear,colorless solution persisted. The organic solution was then added to thepolymer solution while shear mixing at 10,000 RPM for ˜1 minute. Theresulting emulsion, which was a turbid solution, was allowed to stir ina fume hood over night. As the organic solvent evaporated the solutionbecame less turbid and colorless. The next day the solution was filteredthrough a 0.22 micron dead-end filter, and the resulting solution wasthen subjected to ultrafiltration with a Spectrum Labs KrosFlotangential flow filtration system and a 10 kDa diafiltration membrane.The solution was concentrated from 2 mg/mL polymer concentration toapproximately 20 mg/mL polymer concentration, and Iron (III) Chloridewas added to the formulation for a final concentration of 10 mM. The pHof the solution was then adjusted to 7.0 with NaOH and stirred at roomtemperature for 4 hours. The solution was then adjusted to 5 mg/mLpolymer concentration with water, and concentrated to approximately 20mg/mL by ultrafiltration. The solution was then frozen at −40 degreesCelsius and lyophilized.

Example 138

Rat Pharmacokinetics of Daunorubicin Micelles Compared to FreeDaunorubicin

Fisher rats that possessed a jugular vein catheter were injected with 10mg/kg of free daunorubicin, crosslinked (hydroxamic acid) daunorubicinmicelle (prepared according to Example 113), and carboxylic acidcrosslinked daunorubicin loaded micelles (prepared according to Example130) by a fast IV bolus with an injection volume of 2 mL. The deliveryvehicle for drug administration was isotonic saline. Rat blood wascollected from the catheter into K₂-EDTA tubes by heart puncture at timepoints of 1, minute, 5 minutes, 15 minutes, 1 hour, 4 hours, 8 hours and24 hours. Plasma was isolated by centrifugation at 1000 RPM for 5minutes, and 150 uL of extraction solution (ice cold methanol/100 ng/mLdaunorubicin internal standard) was added to 50 uL of each plasmasample. Samples were then vortexed for 10 minutes, centrifuged at 13,000RPM for 10 minutes, and 150 uL of the supernatant is transferred to HPLCvials for analysis. Samples were analyzed on a Waters Alliance 2695equipped with a 2475 fluorescence detector (Ex=470 nm; Em=580). A 5 μLsample injection was made onto a Waters 4 μm Nova Pak C18 (3.9×150 mm)at 30° C. with a flow rate of 0.750 mL per minute of 10 mM phosphatebuffer (pH=1.4), methanol and acetonitrile (gradient from 70/10/20 to40/10/50 for buffer/methanol/acetonitrile was made over eight minutes).Analyte eluted at 5.9 minutes under these conditions, was normalized tothe internal standard, and quantitated using a standard curve comprisedof seven standards. The pharmacokinetic parameters are summarized in thetable below and the curves are shown in FIG. 21. The exposure ofdaunorubicin to the plasma compartment as determined by the area underthe concentration versus time curve (AUC) delivered by the hydroxamicacid formulation was 383.6 μg*h/mL. The terminal (elimination) half-lifeof daunorubicin delivered to the plasma by the formulation was 3.9hours. This is compared to the free drug that showed an AUC of 1.3μg*h/mL and a half life of 3.4 hours as well as the carboxylic acidformulation that showed an AUC of 51.8 μg*h/mL and a half life of 2.4hours. Therefore, the carboxylic acid crosslinked formulations had anexposure of 40 times higher than free drug, and the hydroxamic acidformulation had an exposure of 295 times better than the free drug.

Sample AUC (μg*h/mL) CMax (μg/mL) Half-life (h) Hydroxamic Acid 383.6144.0 3.9 Formulation from Example 113 Carboxylic Acid 51.8 143.5 2.4Formulation from Example 130 Free Daunorubicin 1.3 3.3 3.3

Example 139

Rat Pharmacokinetics of Crosslinked Cabizataxel Micelles

Fisher rats that possessed a jugular vein catheter were injected with 5mg/kg of free cabizataxel or crosslinked cabizataxel micelle (preparedaccording to Example 132) by a fast IV bolus with an injection volume of2 mL. The delivery vehicle for drug administration was isotonic saline.Rat blood was collected from the catheter into K₂-EDTA tubes by heartpuncture at time points of 1, minute, 5 minutes, and 15 minutes. Plasmawas isolated by centrifugation at 1000 RPM for 5 minutes, and 150 uL ofextraction solution was added to 50 uL of each plasma sample. Sampleswere then vortexed for 10 minutes, centrifuged at 13,000 RPM for 10minutes, and 150 uL of the supernatant is transferred to HPLC vials foranalysis. FIG. 22 demonstrates the concentration of cabazitaxel in therat plasma for the first 15 minutes after test article administration.The exposure of cabazitaxel to the plasma compartment over 15 minuteswas 10 μg*h/mL with a CMax of 44.5 μg/mL, compared to 0.2 μg*h/mLexposure for the free drug with a CMax of 1.2 μg/mL.

Example 140

Anti-Tumor Efficacy of SN-38 Micelles

HCT-116 colon cancer cells were cultured according to ATCC guidelines,harvested by trypsin incubation, and resuspended at a concentration of 2million cells per 0.1 mL in saline for injection. Mice were inoculatedby injecting 0.1 mL (i.e. 2 million cells) subcutaneously into the rightflanks of the mice. When tumors reached approximately 100 mm³ the micewere randomized into treatment groups. Each group consisted of 8 miceper group. Treatment groups included saline control; polymer control;free irinotecan at 35 mg/kg; and SN-38 formulation from Example 127C at20, 35, and 50 mg/kg. Mice were dosed by a fast IV bolus into the tailvein; the injection volume was 0.2 mL. Tumors were measured by digitalcaliper, and volume (mm³) was calculated using the formula V=(W²×L)/2,where width (W) is the largest diameter measurement and length (L) isthe diameter measurement perpendicular to the width. The dosing schedulewas once a week for three weeks (3×QW). The vehicle for polymer deliverywas isotonic saline. Clinical observations during the study includedchanges in mouse body weight, morphological observations of sick mousesyndrome (dehydration, spinal curvature, and opportunistic infections ofthe eyes, genitals, or skin rash), and gross pathological changesdetermined by necropsies upon termination of the experiment. The graphof the growth rate is shown in FIG. 23. The data showed a 6-foldincrease in tumor volume for the saline control group, with a meangrowth rate of 46.8 mm³ per day. The polymer control group saw nostatistical difference in tumor growth compared to the saline controlgroup, with a 5.5-fold increase in volume and a mean growth rate of 43.7mm³ per day. The irinotecan at 50 mg/kg free drug control group saw a40% reduction in tumor volume compared to saline, with a 2.7-foldincrease in volume and a mean growth rate of 18.9 mm³ per day. The 20mg/kg SN-38 formulation group saw a 71% inhibition in tumor volumecompared to saline control and a mean growth rate of 13.6 mm³ per day.The 35 mg/kg SN-38 formulation group saw 30% regression in tumor volumewith a 1.5 fold decrease in size and a mean tumor regression rate of−2.4 mm³ per day. The 50 mg/kg SN-38 formulation group saw 47.6%regression in tumor volume with a 2.1 fold decrease in size and a meantumor regression rate of −3.8 mm³ per day.

Example 141

Pharmacokinetics and Biodistribution of Crosslinked Aminopterin Micelles

Female athymic nude mice were supplied by Harlan (Indianapolis, Ind.).Mice were received at 4-5 weeks of age, 12-15 g in weight. The mice werehoused in microisolator and maintained under specific pathogen-freeconditions. Study Female mice were inoculated subcutaneously in theright flank with 0.1 ml of a 50% RPMI/50% Matrigel™ (BD Biosciences,Bedford, Mass.) mixture containing a suspension of OVCAR-3 tumor cells(approximately 5.0×106 cells/mouse). Tumors were measured using calipersand tumor weight was calculated using the formula V=(W²×L)/2, wherewidth (W) is the largest diameter measurement and length (L) is thediameter measurement perpendicular to the width. Study start days werestaggered by group due to varying growth patterns in the tumors. Animalswere administered test material, aminopertin micelles from Example 131at 20 mg/kg, once tumor volume reached 150-250 mm³. Upon euthanizationof each mouse at 5 and 15 minutes, 1, 4, 12, 24, and 48 hours aftertreatment (4 mice per timepoint), plasma, tumor, spleen, liver and lungspecimens were collected. Heparinized mouse plasma and tissue samples(liver, lung, spleen and tumor) were analyzed using a high pressureliquid chromatography assay with tandem mass spectral detection(LC-MS/MS). Calibrator and quality control (QC) samples were prepared byspiking aminopterin into sodium heparinized human plasma. Tissue sampleswere homogenized in 50% methanol and stored frozen at −80° C. untilanalyzed. Each study matrix type was analyzed in a separate analyticalbatch along with duplicate calibration and QC samples. A 100 μl aliquotof the calibrator, QC, blank, or study sample (plasma or tissuehomogenate) was mixed with 50.0 μL of dilution buffer (1.0 mM ammoniumformate containing 0.1% formic acid) followed by 400 μL of acetonitrilecontaining the internal standard (IS; methotrexate 50.0 ng/ml) in amicrocentrifuge tube to precipitate proteins. The tubes were capped,vortexed, allowed to digest for 5 minutes, and centrifuged at 14,000 rpmand 4° C. for 5 minutes. A 100 uL aliquot of the supernatant was dilutedwith 1.5 mL of dilution buffer, vortex mixed, and 20 uL injected intothe LC-MS/MS system. The concentration of each sample was determined bycomparison to a standard curve. Concentration-time curves for eachcompartment were constructed and pharmacokinetic data calculated foreach compartment. The mean plasma and tissue PK profiles can be seen inFIG. 24. Plasma NCA determined the mean half-life of Aminopterin to be37.65 hours. The mean AUC0-48 hr in plasma was found to be 12571ng*hr/ml. The mean half-life of Aminopterin in tumor, lungs and spleenwas determined to be 9.65, 11153 and 51.87 hours, respectively. Theterminal slope of the liver concentrations did not allow for a half-lifecalculation since at 48 hours the concentration was higher than at 12and 24 hours. The mean AUC0-48 hr of tumor, lungs, spleen and liver wasfound to be 9559, 4276, 4586, and 9909 ng*hr/g, respectively.

Example 142

Anti-Tumor Efficacy of Crosslinked Aminopterin Micelles

The MFE-296 human endometrial tumor cell line was received from andcultured according to ATCC. Female athymic NCR nude mice(CrTac:NCr-Foxnlnu) were supplied by Taconic. Female athymic nude micewere inoculated subcutaneously in the right flank with 0.1 ml of a 50%RPMI 1640/50% Matrigel™ (BD Biosciences, Bedford, Mass.) mixturecontaining a suspension of MFE-296 tumor cells (approximately 1×10⁷cells/mouse). Twenty days following inoculation, tumors were measuredusing calipers and tumor weight was calculated using the formulaV=(W²×L)/2, where width (W) is the largest diameter measurement andlength (L) is the diameter measurement perpendicular to the width. Fiftymice with tumor sizes of 80-257 mm³ were randomized into five groups often mice each with a mean of approximately 143 mm³ by randomequilibration. Body weights were recorded when the mice were randomizedand were taken twice per week thereafter in conjunction with tumormeasurements. Treatment groups included polymer control, freeaminopterin at 1.5 mg/kg, aminopterin micelles from Example 132 at 1.5mg/kg and 7.5 mg/kg. Treatments were performed on Day 1, 8, and 15, oronce a week for three weeks (3×QW) by tail vein intravenousadministration. Injections were 0.2 mL and the vehicle was isotonicsaline. The graph of the tumor growth for each group is shown in FIG.25. The polymer control group reached a mean tumor weight of 973.9 mg byDay 28. This group experienced no appreciable body weight loss duringthe study. No adverse dosing reactions were observed. Treatment withaminopterin formulation at 1.5 mg/kg resulted in a mean tumor weight of1330.4 mg by Day 28. This group produced no reportable inhibition whencompared to the vehicle control on Day 28. This group experienced noappreciable body weight loss during the study. No adverse dosingreactions were observed. Treatment with aminopterin formulation 7.5mg/kg resulted in a mean tumor weight of 599.7 mg by Day 28. This groupproduced an inhibition of 44.8% when compared to the vehicle control onDay 28. This group experienced mild body weight loss with a maximum of4.3% on Day 4. Body weights were fully recovered by Day 15. No adversedosing reactions were observed. Treatment with free aminopterin 1.5mg/kg resulted in a mean tumor weight of 1115.1 mg by Day 28. This groupproduced no reportable inhibition when compared to the vehicle controlon Day 28. No significant difference in tumor weight was observed whencompared to the vehicle control on Day 28. This group experienced noappreciable body weight loss during the study.

We claim:
 1. A triblock copolymer of the following structure:

n is 20-500; m is 0, 1, or 2; x is 3 to 50; y is 5 to 100; each R^(y) isindependently selected from one or more of an aspartic acid side chaingroup, a leucine side chain group, a phenylalanine side chain group, anda tyrosine side chain group; R¹ is —Z(CH₂CH₂Y)_(p)(CH₂)_(t)R³, wherein:Z is —O—; Y is —O—; p is 0-10; t is 0-10; and R³ is —CH₃ or


2. The triblock copolymer of claim 1, wherein R^(y) is a mixture ofleucine and tyrosine side chain groups.
 3. The triblock copolymer ofclaim 1, wherein R^(y) is a mixture of phenylalanine and tyrosine sidechain groups.
 4. The triblock copolymer of claim 1, wherein R^(y) is amixture of leucine, aspartic acid, and tyrosine side chain groups. 5.The triblock copolymer of claim 1, wherein R^(y) is a mixture ofphenylalanine, aspartic acid, and tyrosine side chain groups.
 6. Thetriblock copolymer of claim 1, of the following structure:


7. The triblock copolymer of claim 1, of the following structure:


8. The triblock copolymer of claim 1, of the following structure: